Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases Abstract Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn2+, the manganese peroxidase (MnP) substrate (Mn3+ being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these ‘hybrid’ properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn2+ oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not β-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed. Crystal structures, fungal peroxidases, haem access-channel, lignin biodegradation, manganese-binding site, site-directed mutagenesis, spectroscopic analyses, transient-state kinetics, tryptophanyl radical, versatile peroxidase Ligninolytic peroxidases Degradation of lignin is a key process for carbon recycling in land ecosystems, as well as for utilization of lignocellulosic biomass in paper pulp manufacture and bioethanol production. Degradation of this recalcitrant aromatic polymer is caused in nature by wood-rotting fungi through a process that was defined as an enzymatic combustion (Kirk and Farrell, 1987). Several oxidoreductases (including peroxidases, oxidases, and laccases) secreted by ligninolytic basidiomycetes, the so-called white-rot fungi, are involved in lignin biodegradation (Higuchi, 2004; Martínez et al., 2005; Kersten and Cullen, 2007). Ligninolytic haem peroxidases are able to break down model compounds representative of the main linkage types in lignin due to their high redox-potential and specialized catalytic mechanisms (Martínez, 2002, 2007; Hammel and Cullen, 2008). Extracellular oxidases, such as glyoxal oxidase (a copper-radical protein) (Kersten and Kirk, 1987) and aryl-alcohol oxidase (a flavoprotein) (Guillén and Grans, 1994), provide the hydrogen peroxide required by peroxidases. Finally, laccases can directly oxidize only minor phenolic lignin moieties, although their biotechnological interest has increased after discovering that redox mediators (Bourbonnais and Paice, 1990), including natural phenols (Camarero et al., 2005), strongly enhance their oxidizing power. Recently, an extracellular haem-thiolate peroxidase, which is also able to degrade lignin model compounds, among other enzymatic reactions, has been found in soil Agaricales, such as Agrocybe aegerita (V Briganti) Singer and several Coprinus species (Hofrichter and Ullrich, 2006). Ligninolytic haem peroxidases from white-rot fungi include lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (Gold et al., 2000; Martínez, 2002; Hammel et al., 2008). Due to the high redox-potential required for lignin oxidation, ligninolytic peroxidases have been the object of high interest as industrial biocatalysts (Martínez, 2007). LiP and MnP were first described in Phanerochaete chrysosporium Burdsall (order Corticiales) and genes/cDNA encoding isoenzymes LiPH8 and MnP1 cloned in 1987–1988 from the same fungus (Cullen and Kersten, 2004). From this date, related genes have been cloned from other Corticiales, Agaricales, and Polyporales basidiomycetes including: (i) over 20 different mnp genes from Agaricus bisporus (J.E. Lange) Imbach, Ceriporiopsis rivulosa (Berk. & M.A. Curtis) Gilbertson & Ryvarden, Ceriporiopsis subvermispora (Pilat) Gilbertson & Ryvarden, Dichomitus squalens (P. Karsten) D.A. Reid, Lentinula edodes (Berk.) Pegler, Phlebia radiata Fries, P. chrysosporium, Phanerochaete sordida (P. Karsten) J. Eriksson & Ryvarden, Pleurotus ostreatus (Jacquin) P. Kummer, and Trametes versicolor (L.) Lloyd; and (ii) around 15 lip genes from Bjerkandera adusta (Willd.) P. Karst., P. radiata, P. chrysosporium, Trametes cervina (Schwein.) Bres., and T. versicolor (Ruiz-Dueñas et al., 2007a). By contrast, VP genes have been cloned only from a few species from the genera Pleurotus (order Agaricales), including Pleurotus eryngii (DC.) Gillet and Pleurotus sapidus (Schulzer) Sacc. among others, and Bjerkandera (order Polyporales), including Bjerkandera adusta (Willd.) P. Karst. (Camarero et al., 1999; Ruiz-Dueñas et al., 1999, 2007a; Moreira et al., 2005). VP could also be produced by other Polyporales basidiomycetes including species from the genera Panus (Lisov et al., 2007), Trametes (Ruiz-Dueñas et al., 2007a), and Spongipellis (GenBank AB244274). The evolutionary relationships of all the proteins encoded by these genes are shown in the dendrogram of Fig. 1, which also indicates the presence in the different sequences of residues forming the substrate oxidation sites discussed below (involved in the oxidation of Mn2+ and high redox-potential aromatic substrates). The dendrogram obtained from comparison of the 56 basidiomycete peroxidases available showed a well-defined cluster corresponding to typical MnP (from Phanerochaete, Ceriporiopsis, Phlebia, and Dichomitus). The second cluster is large and includes a small subcluster containing three atypical MnP from Ganoderma species (and the so-called Mn-repressed peroxidase from T. versicolor), and a large subcluster where all LiP and VP, and some MnP (from Pleurotus, Trametes, Phlebia, Lentinula, and Agaricus), are located (together with unclassified peroxidases, such as Trametes PGV and PGVII). In this heterogenous cluster, typical LiP (from Phanerochaete, Bjerkandera, Phlebia, and Trametes) form a relatively homogeneous group, whereas the group of typical VP (from Pleurotus) is small, and other VP are related to different MnP. Finally, two peroxidases from the non-lignicolous fungi Coprinopsis cinerea (Schaeff.) Redhead, Vilgalys and Moncalvo (synonym Coprinus cinereus (Schaeff.) Gray) and Coprinellus disseminatus (Pers.) Gray, and the NOPA peroxidase identified in the P. chrysosporium genome (Larrondo et al., 2005), are not grouped together suggesting that they are not related to ligninolytic peroxidases. Gene fragments from the above peroxidase families have been recently identified in a variety of other basidiomycetes indicating a wider distribution (Morgenstern et al., 2008). Fig. 1. View largeDownload slide Evolutionary relationships and structural–functional classification of basidiomycete peroxidases (mature proteins) including 53 ligninolytic enzymes belonging to the MnP, LiP, and VP families (and some related unclassified peroxidases) and three low-redox potential peroxidases. The phylogenetic analysis was performed using MEGA4 (Tamura et al., 2007) with Poisson-corrected evolutionary distances, complete deletion option (269 positions in the final dataset), and UPGMA clustering method. The structural–functional classification was based on the presence in the protein sequence or molecular structure of the structural motifs characteristic of the above families, namely: (i) Mn2+-oxidation site formed by three acidic residues (red circles); and (ii) exposed catalytic tryptophan oxidizing high redox-potential substrates (blue circles). As a result of the structural analysis, three putative LiP (previously described as MnP) and six putative VP (unclassified or described as LiP or MnP) were identified. The presence of a catalytic tyrosine in the LiP from T. cervina, playing the same function of the above catalytic tryptophan, is also indicated (blue square). Species abbreviations: AB, A. bisporus; B, Bjerkandera sp; BA, B. adusta; CC, C. cinerea; CD, C. disseminatus; CR, C. rivulosa; CS, C. subvermispora; DS, D. squalens; GA, G. applanatum; GAU, G. australe; GF, G. formosanum; IZU, unidentified basidiomycete IZU-154; LE, L. edodes; PC, P. chrysosporium; PE, P. eryngii; PO, P. ostreatus; PP, P. pulmonarius; PR, P. radiata; PS, P. sordida; PSA, P. sapidus; S, Spongipellis sp; TC, T. cervina; and TV, T. versicolor. Protein sequence entries: AB-MnP, CAG27835; B-VP, AAO47909; BA-LiP, 1906181A; CD-PerO, AAZ14938; CC-CIP, CAA50060; CR-MnPA, ABB83812; CR-MnPB, ABB83813; CS-MnP1, AAB03480; CS-MnP2, AAD43581; CS-MnP3, AAD45725; CS-MnP4, AAO61784; DS-MnP1, AAF31329; DS-MnP2, AAF31330; GA-MnP1, BAA88392; GAU-MnP, ABB77244; GF-MnP, ABB77243; IZU-MnP1, no entry available but sequence taken from the literature (Matsubara et al., 1996); LE-MnP1, BAE79199; PC-LiPA (isoenzyme H8), AAA53109; PC-LiPB (isoenzyme H8), AAA33741; PCLiPC (isoenzyme H10), AAA33739; PC-LiPD (isoenzyme H2), CAA33621; PC-LiPE (isoenzyme H8), AAA33738; PC-LiPF, AAA33736; PC-LiPH (isoenzyme H8), AAA56852; PC-LiPJ, AAD46494; PC-MnP1, AAA33744; PCMnP2, AAA33745; PC-MnP3, AAB39652; PC-NOPA, AAU82081; PE-VPL1, AAD01401; PE-VPL2, AAD01404; PE-VPL3, CAD56164 (and DQ056374); PE-VPS1, AAD54310; PO-MnP1, AAA84396; PO-MnP2, CAB51617; PO-MnP3, BAA33449; PP-MnP5, AAX40734; PR-LiP, P20010; PR-LiP1, AAW59419; PR-LiP4, AAW66483; PR-MnP2, CAC85963; PR-MnP3, CAC84573; PS-MnP1, BAC06185; PS-MnP2, BAC06186; PS-MnP3, BAC06187; PSA-VP, CAJ01576; S-MnP1, BAE79812; TC-LiP, AB191466; TV-LiP12, AAA34049; TV-LiP7, CAA83147; TV-LiPGII, CAA53333; TV-MnP2, CAA83148; TV-MRP, AAB63460; TV-PGV, CAA54398; and TV-PGVII, CAA91043. Fig. 1. View largeDownload slide Evolutionary relationships and structural–functional classification of basidiomycete peroxidases (mature proteins) including 53 ligninolytic enzymes belonging to the MnP, LiP, and VP families (and some related unclassified peroxidases) and three low-redox potential peroxidases. The phylogenetic analysis was performed using MEGA4 (Tamura et al., 2007) with Poisson-corrected evolutionary distances, complete deletion option (269 positions in the final dataset), and UPGMA clustering method. The structural–functional classification was based on the presence in the protein sequence or molecular structure of the structural motifs characteristic of the above families, namely: (i) Mn2+-oxidation site formed by three acidic residues (red circles); and (ii) exposed catalytic tryptophan oxidizing high redox-potential substrates (blue circles). As a result of the structural analysis, three putative LiP (previously described as MnP) and six putative VP (unclassified or described as LiP or MnP) were identified. The presence of a catalytic tyrosine in the LiP from T. cervina, playing the same function of the above catalytic tryptophan, is also indicated (blue square). Species abbreviations: AB, A. bisporus; B, Bjerkandera sp; BA, B. adusta; CC, C. cinerea; CD, C. disseminatus; CR, C. rivulosa; CS, C. subvermispora; DS, D. squalens; GA, G. applanatum; GAU, G. australe; GF, G. formosanum; IZU, unidentified basidiomycete IZU-154; LE, L. edodes; PC, P. chrysosporium; PE, P. eryngii; PO, P. ostreatus; PP, P. pulmonarius; PR, P. radiata; PS, P. sordida; PSA, P. sapidus; S, Spongipellis sp; TC, T. cervina; and TV, T. versicolor. Protein sequence entries: AB-MnP, CAG27835; B-VP, AAO47909; BA-LiP, 1906181A; CD-PerO, AAZ14938; CC-CIP, CAA50060; CR-MnPA, ABB83812; CR-MnPB, ABB83813; CS-MnP1, AAB03480; CS-MnP2, AAD43581; CS-MnP3, AAD45725; CS-MnP4, AAO61784; DS-MnP1, AAF31329; DS-MnP2, AAF31330; GA-MnP1, BAA88392; GAU-MnP, ABB77244; GF-MnP, ABB77243; IZU-MnP1, no entry available but sequence taken from the literature (Matsubara et al., 1996); LE-MnP1, BAE79199; PC-LiPA (isoenzyme H8), AAA53109; PC-LiPB (isoenzyme H8), AAA33741; PCLiPC (isoenzyme H10), AAA33739; PC-LiPD (isoenzyme H2), CAA33621; PC-LiPE (isoenzyme H8), AAA33738; PC-LiPF, AAA33736; PC-LiPH (isoenzyme H8), AAA56852; PC-LiPJ, AAD46494; PC-MnP1, AAA33744; PCMnP2, AAA33745; PC-MnP3, AAB39652; PC-NOPA, AAU82081; PE-VPL1, AAD01401; PE-VPL2, AAD01404; PE-VPL3, CAD56164 (and DQ056374); PE-VPS1, AAD54310; PO-MnP1, AAA84396; PO-MnP2, CAB51617; PO-MnP3, BAA33449; PP-MnP5, AAX40734; PR-LiP, P20010; PR-LiP1, AAW59419; PR-LiP4, AAW66483; PR-MnP2, CAC85963; PR-MnP3, CAC84573; PS-MnP1, BAC06185; PS-MnP2, BAC06186; PS-MnP3, BAC06187; PSA-VP, CAJ01576; S-MnP1, BAE79812; TC-LiP, AB191466; TV-LiP12, AAA34049; TV-LiP7, CAA83147; TV-LiPGII, CAA53333; TV-MnP2, CAA83148; TV-MRP, AAB63460; TV-PGV, CAA54398; and TV-PGVII, CAA91043. Versatile peroxidase VP oxidizes Mn2+, as MnP (EC 1.11.1.13) does, and also high redox-potential aromatic compounds, as LiP (EC 1.11.1.14) does. Due to their Mn-oxidizing activity, the Pleurotus VP isoenzymes were first described as MnP isoenzymes (Martínez et al., 1996; Giardina et al., 2000), but they were later recognized as representing a new peroxidase type (EC 1.11.1.16). VP is also able to efficiently oxidize phenolic compounds and dyes that are the substrates of generic peroxidases (EC 1.11.1.7), such as the C. cinerea peroxidase (CIP) (Baunsgaard et al., 1993) and related peroxidases, or the well-known horseradish peroxidase (HRP) (Veitch, 2004). By contrast, LiP is not able to oxidize phenolic compounds efficiently because of inactivation in the absence of veratryl alcohol (VA) or related substrates, and MnP only oxidize phenols in the presence of Mn2+, although a P. radiata short MnP seems to be an exception (Hildén et al., 2005). Moreover, VP directly oxidizes high redox-potential compounds, for example, the dye Reactive Black 5 (RB5), that LiP can oxidize only in the presence of redox mediators such as VA (Heinfling et al., 1998b). The Mn3+ formed by VP (and MnP) acts as a diffusible oxidizer on phenolic lignin and free phenols, as well as a starter of lipid peroxidation reactions involved in the biodegradation of recalcitrant compounds (Kapich et al., 2005). The VP family is included in Class II (fungal peroxidases) of the ‘plant, fungal, and bacterial peroxidase’ superfamily (Welinder, 1992) together with the MnP, LiP, and CIP families. The interest on VP has increased during the last years, both as a model enzyme and as a source of industrial/environmental biocatalysts. The molecular architecture of VP and other basidiomycete peroxidases includes a haem cofactor located at an internal cavity connected to the solvent by two access channels (Gold et al., 2000; Martínez, 2002). The main channel is conserved in all haem peroxidases and is used by hydrogen peroxide to reach and oxidize the haem cofactor forming the two-electron activated Compound I, which is stepwise reduced to Compound II and resting enzyme during two one-electron substrate oxidations. The second channel extends directly to the haem propionates being the site where MnP and VP oxidize Mn2+ to Mn3+, as described below. Bifunctional prokaryotic (Class I) ascorbate peroxidase also oxidizes ascorbate near the haem propionates (Sharp et al., 2003). Oxidation of phenolic compounds has been studied in peroxidases from Class III (plant peroxidases) and also from Class II, and the classical hypothesis that the reaction takes place in the vicinity of the haem has been demonstrated for plant HRP and fungal CIP (Smith and Veitch, 1998). However, no crystal structure of a ligninolytic peroxidase complexed with an aromatic substrate or analogue has been reported to date. Moreover, it is generally accepted that the main haem channel, in spite of certain degree of plasticity (Gerini et al., 2003), is too narrow for direct contact between the haem cofactor and most aromatic substrates including lignin. Therefore, long-range electron transfer (LRET) from a protein radical at the surface of the enzyme, which would act as the substrate oxidizer, to the haem cofactor has been suggested to explain oxidation of aromatic substrates, redox mediators, and even the lignin polymer. As discussed below, dual oxidation of some aromatic substrates and dyes at the VP surface (by LRET) and at the haem channel is considered in the light of some recent results. Mn-oxidation site in VP Enzymatic oxidation of Mn2+ to Mn3+ is a unique characteristic of basidiomycete MnP and VP, although it has also been reported in prokaryotic catalase-peroxidase (Magliozzo and Marcinkeviciene, 1997). The Mn2+-oxidation site in P. eryngii VP is similar to that of P. chrysosporium MnP (Gold et al., 2000; Sundaramoorthy et al., 2005), but it has certain differences as revealed by crystallographic, site-directed mutagenesis, and kinetic studies (Ruiz-Dueñas et al., 2007b). The Mn2+-binding site in VP is formed by the side-chains of Glu36, Glu40, and Asp175 located in front of the internal (i.e. more distant from the main haem access-channel) propionate of haem (Fig. 2, top), and connected to the solvent by a narrow access-channel that presents a variable geometry during catalysis. The carboxylates of the above amino-acid residues and the haem propionate are responsible of Mn2+ binding for subsequent electron transfer to the activated haem of VP Compounds I and II. Fig. 2. View largeDownload slide Axial views of the haem region showing the exposed catalytic tryptophan in VP (Trp164) and LiP (Trp171) responsible for oxidation of high redox-potential substrates by LRET to haem (red arrow), and the residues forming the Mn2+-binding site near the internal propionate of haem in VP (Glu36, Glu40, and Asp175) and MnP (Glu35, Glu39, and Asp179). Mn2+ is shown as a van der Waals sphere, and several water molecules are included (red spheres). From PDB entries, 2BOQ, 1YYD, and 1LLP for VP, MnP, and LiP, respectively. Fig. 2. View largeDownload slide Axial views of the haem region showing the exposed catalytic tryptophan in VP (Trp164) and LiP (Trp171) responsible for oxidation of high redox-potential substrates by LRET to haem (red arrow), and the residues forming the Mn2+-binding site near the internal propionate of haem in VP (Glu36, Glu40, and Asp175) and MnP (Glu35, Glu39, and Asp179). Mn2+ is shown as a van der Waals sphere, and several water molecules are included (red spheres). From PDB entries, 2BOQ, 1YYD, and 1LLP for VP, MnP, and LiP, respectively. The crystal structures (solved up to 1.3 Å resolution) of wild and Escherichia coli (Migula) Castellani & Chalmers expressed VP showed a variable orientation of the Glu36 and Glu40 side-chains, whereas that of Asp175 is maintained (Ruiz-Dueñas et al., 2007b). The position in the in vitro activated recombinant VP (Pérez-Boada et al., 2002) before its exposure to Mn2+ (and in the Mn2+ exposed D175A variant, which suffer from a strong decrease in the ability to bind and oxidize the cation) corresponds to an ‘open-gate’ conformation enabling Mn2+ access to the oxidation site. The conformation in which the two glutamate side-chains point toward the Mn2+ ion correspond to the ‘closed-gate’ conformation, and was found in the naturally-folded enzyme from Mn2+-containing P. eryngii cultures. In this conformation Glu36, Glu40, Asp175, and propionate carboxylates are at co-ordination distance from Mn2+. In contrast to that reported for MnP (Sundaramoorthy et al., 1994, 1997), the recombinant VP crystals grown in the presence of Mn2+ were unstable, and the addition of Zn2+ to grow crystals after Mn2+ exposure resulted in an intermediate conformation of the metal binding site, with the Glu40 side-chain at a position different from that found in wild VP crystals. The ‘closed-gate’ conformation described above coincides with that observed in P. chrysosporium MnP crystallized in the presence of Mn2+, where three homologous residues (Glu35, Glu39, and Asp179) participate in metal co-ordination (Sundaramoorthy et al., 1994) (Fig. 2, centre). Opening of the glutamate side-chains was not observed in MnP crystals grown in the absence of Mn2+ (Sundaramoorthy et al., 1997). However, displacement of Glu35 and Glu39 side-chains was found in crystals of a mutated MnP variant that does not bind Mn2+ (Sundaramoorthy et al., 1997) and in MnP crystals where the inhibitor Sm3+ was removed from the metal binding site (Sundaramoorthy et al., 2005). Additional amino acid residues, which are absent in VP, contribute to orientation of the Mn2+ ligands in MnP affecting their mobility. This is the case of Arg177 forming a salt bridge with Glu35 necessary for the optimal Mn2+ co-ordination and ligation geometry in MnP (Gelpke et al., 2000) Site-directed mutagenesis was performed to evaluate the involvement of the three above acidic residues in VP catalysis (Ruiz-Dueñas et al., 2007b). The E36A, E40A, and D175A mutations caused 4–20-fold decrease in Mn2+ oxidation activity (kcat) under steady-state conditions (characterized by VP saturating concentrations of both oxidizing and reducing substrates). Moreover, the selectivity constant (kcat/Km, also known as catalytic efficiency) was 250–1250-fold decreased, indicating that the above residues are responsible for selective binding of Mn2+ near the haem. The decreases in VP selectivity constant were much lower than found in similar MnP variants (E40A and D175A) (Kishi et al., 1996; Whitwam et al., 1997; Youngs et al., 2001). This is due to differences in kcat, which decreased from similar values in both native enzymes (around 300 s−1) to much lower values in the MnP variants (1–3 s−1) than in the VP variants (15–85 s−1). In fact, removal of the three carboxylates in a triple variant (E36A/E40A/D175A) was required to lower the VP activity to levels (<1% kcat) similar to those found in MnP single variants. Transient-state kinetic constants showed that reduction of both VP Compounds I and II by Mn2+ (estimated from changes of their characteristic spectra using stopped-flow rapid spectrophotometry) was modified by the E36A, E40A, and D175A mutations. The apparent second-order rate constants revealed that reduction of Compound II was much more affected (103–104-fold lower k3app) than Compound I reduction (80–325-fold lower k2app), the former being a rate-limiting step in VP catalysis. Site-directed mutagenesis of P. chrysosporium MnP also showed that reduction of both Compound II (Kusters-van Someren et al., 1995; Kishi et al., 1996) and Compound I (Whitwam et al., 1997; Youngs et al., 2001) by Mn2+ was lowered by mutations at the binding site. However, the decreases of apparent second-order rate constants were lower in the VP than in the homologous MnP variants. Moreover, the first-order constants for Compound II reduction (Compound I reduction is too fast to be measured) showed a strong increase of the dissociation constant (300–1000-fold), but only slightly affected k3 (2–30-fold decrease) in the case of VP mutants, whereas a strong decrease in k3 (100–1200-fold) was also observed in the corresponding MnP variants. The selectivity constant for Mn2+ also decreased (50–75-fold) when the acidic side-chains were shortened in the E36D and E40D variants that, nevertheless, retained 30–50% of their steady-state activity (kcat). This strongly contrasts with the behaviour of P. chrysosporium MnP, where similar mutations (E35D and E39D) caused 50–100 fold decrease of kcat (Kishi et al., 1996; Youngs et al., 2001). The same tendency was observed in the first-order rate constant for Compound II reduction (k3) that showed 40–120-fold lower values in the MnP variants but only a 1–4-fold decrease in the VP variants. The kinetic constants of VP mutated variants at Glu36 and Glu40 positions showed that the Mn2+-oxidation site of VP, although having the same Mn-co-ordinating residues as found in MnP, operates in a slightly different way, in the sense that Mn2+ is still efficiently oxidized when one of the three amino acid carboxylates is removed (Glu/Asp→Ala mutations) or when the length of the acidic side-chains is reduced (Glu→Asp mutations) resulting in much higher kcat and k3 values than found for homologous MnP variants. A putative MnP has been reported in Ganoderma applanatum (Pers.) Pat., Ganoderma australe (Fr.) Pat., and Ganoderma formosanum T.T. Chang & T. Chen (Maeda et al., 2001) with only two acidic residues at the putative Mn2+-oxidation site. However, the catalytic properties of these peroxidases should be re-examined, since sequence alignment showed a tryptophan residue homologous to the VP Trp164 (or LiP Trp171) discussed below (Fig. 1). Mn-oxidation sites (red circles in Fig. 1) have been identified in the sequences (and molecular models when available) of 23 MnP and six VP, as well as in five putative VP that were initially described as MnP, including MnP1 from P. ostreatus and MnP5 from Pleurotus pulmonarius (Fr.) Quel., or LiP (and one of them as an unclassified Mn-repressed peroxidase). The kinetic constants of these putative VP on representative substrates should be re-examined. VP oxidation of high redox-potential substrates: LRET mechanism LRET occurs in different redox proteins, like prokaryotic cytochrome-c peroxidase that oxidizes cytochrome c at the protein surface, and transfers electrons to a stable tryptophan radical near the haem cofactor via three amino acid residues (Pelletier and Kraut, 1992). LRET has been suggested to be involved in lignin biodegradation by ligninolytic haem peroxidases for electron transfer both: (i) in the peroxidase molecule, to overcome the steric hindrances preventing direct interaction between the haem group and the lignin polymer; and (ii) in the lignin macromolecule itself resulting in breakdown of the most labile inter-unit linkages (Schoemaker et al., 1994a, b). Crystal structures and homology models of two P. eryngii VP isoenzymes revealed three possible LRET pathways for the oxidation of high redox-potential aromatic compounds (Camarero et al., 1999; Ruiz-Dueñas et al., 1999; Pérez-Boada et al., 2005). They would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 (homologous to VPL Trp164) of isoenzyme VPS1. These residues are exposed, and less than 11 Å apart from the haem. The corresponding pathways would be similar to those proposed for P. chrysosporium LiP that would start at His82, Trp171, or His239 (Du et al., 1992; Schoemaker et al., 1994a; English et al., 1995; Johjima et al., 1999). With the purpose of investigating the functionality of these three putative pathways, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways present in this isoenzyme; and (ii) incorporated the absent pathway (Pérez-Boada et al., 2005). The steady-state kinetic constants of the variants obtained definitively showed that Trp164 (see Fig. 2, top) is required for the oxidation of the two high redox-potential model substrates investigated, VA and RB5, whereas the two other putative pathways (starting at His232 and His82) did not seem to be involved in catalysis. Transient-state kinetics showed that the W164S mutation completely blocked the reduction of Compound II by both substrates, whereas the apparent second-order rate constant for Compound I reduction was only partially decreased (15–50-fold lower k2app values) (Ruiz-Dueñas et al., 2008a). The above results were in agreement with those obtained by mutagenesis of homologous Trp171 (Fig. 2, bottom) and His82 in P. chrysosporium LiP (mutagenesis of His239 has not been reported) (Doyle et al., 1998; Mester et al., 2001a; Gelpke et al., 2002) and contrasted with some unconfirmed results suggesting that VA could be oxidized at the haem channel (Ambert-Balay et al., 1998). Involvement of a tryptophan residue in VP catalysis had been suggested by N-bromosuccinimide bromination of Pleurotus and Bjerkandera VP (Ayala-Aceves et al., 2001; Ruiz-Dueñas et al., 2001; Kamitsuji et al., 2005; Pogni et al., 2005). In addition to the site-directed mutagenesis of P. eryngii VP described above, mutagenesis of the catalytic tryptophan of P. ostreatus VP has recently been reported using a homologous expression system (Tsukihara et al., 2008). It has also been shown that a tetrameric lignin model compound is degraded by LiP but it is not oxidized by its W171S variant (Mester et al., 2001a). This suggests that polymeric lignin would be oxidized at the same exposed tryptophan involved in VA oxidation. However, this conclusion requires additional investigation, since it has also been reported that a lignin dehydrogenation polymer would bind LiP at His239 (Johjima et al., 1999). Concerning the aromatic substrate oxidation mechanism, Pérez-Boada et al. (2005) provided the first direct evidence on the formation of a protein (Trp164) radical in a ligninolytic peroxidase using low-temperature electron paramagnetic resonance (EPR) of H2O2-activated P. eryngii VP. However, indirect evidence on the formation of a Trp171 radical had already been reported in LiP (Blodig et al., 1999). This is in agreement with EPR results obtained with B. adusta VP (Pogni et al., 2005), but contrasted with unconfirmed LiP studies that assigned the Compound I EPR signal to a porphyrin π cation radical (Khindaria and Aust, 1996). Additional spectroscopic and density functional theory studies confirmed the location of the tryptophan radical in VP, and showed that it exists in the neutral form (Pogni et al., 2006, 2007). Substitution of Trp164 either by serine or histidine resulted in similar incapability of both variants to oxidize high redox-potential compounds, indicating that the imidazole side-chain is not playing a similar role as the indole side-chain. Interestingly, a LiP-type peroxidase has been reported in T. cervina that has an exposed tyrosine residue involved in catalysis, although located at a different position than the LiP and VP catalytic tryptophan (Miki et al., 2006). However, the W164Y variant of VP was not catalytically-active on VA and RB5 under steady-state conditions due to the blocked reduction of Compound II, although the corresponding tyrosinyl radical was clearly detected by EPR (Ruiz-Dueñas et al., 2008c). A significant difference between VP and P. chrysosporium LiP concerns autocatalytic modification of LiP Trp171. It has been reported that Trp171 in P. chrysosporium LiP is naturally β-hydroxylated, most probably due to peroxide excess (with respect to reducing substrate) in fungal cultures (Blodig et al., 1998, 2001). However, this is not the case in VP as shown by X-ray diffraction of crystals from both wild and peroxide-treated recombinant VP (Pérez-Boada et al., 2005). The origin and physiological significance of this difference is to be determined. Exposed tryptophan residues homologous to VP Trp164 (blue circles in Fig. 1) have been identified in the sequences (and molecular models when available) of 14 LiP and six VP, as well as in three putative LiP and five putative VP, the latter already mentioned above. The kinetic constants of these putative LiP (and VP) on veratryl alcohol and other substrates should be re-examined. VP oxidation of high redox-potential substrates: catalytic Trp164 environment Some characteristics of the surface environment of the catalytic tryptophan (VP Trp164 and LiP Trp171) could also be at the origin of differences in the oxidation of high redox-potential aromatic substrates, such as the need for redox mediators in LiP oxidation of some compounds that are directly oxidized by VP (e.g. RB5), and the lower VP efficiency oxidizing VA. With the purpose of investigating this hypothesis, different residues near the Trp164 of VP were modified by site-directed mutagenesis (Ruiz-Dueñas et al., 2008a). Some of the mutations strongly modified the kinetics of VA and RB5 oxidation by VP, demonstrating that other residues at the catalytic tryptophan environment contribute to substrate oxidation by ligninolytic peroxidases. The most outstanding characteristic of the LiP surface around the catalytic Trp171 is the protruding side-chain of Phe267 in an environment with a partial negative charge, whereas VP has several basic residues in the Trp164 environment, and Ala260 at the position of LiP Phe267 (Ruiz-Dueñas et al., 2008a). It was first considered by the authors that the lack of direct activity of LiP on RB5 could be due to the presence of Phe267 interrupting the furrow where this large substrate would be accommodated in VP. However, this did not seem to be the case since introduction of a phenylalanine residue (A260F variant) did not modify RB5 oxidation by VP. By contrast, VP activity on RB5 was eliminated by the R257D mutation. Therefore, the current hypothesis is that the lack of LiP activity on RB5 would not be due to the steric hindrances mentioned above, and also suggested by other authors (Tsukihara et al., 2008), but to the existence of a partially-negative environment (including Asp264 homologous to VP Arg257, and Asp165 homologous to VP Ser158) compared with the positively-charged environment of VP Trp164 (including Lys264 in addition to Arg257) that would favour binding of anionic substrates. On the other hand, it was demonstrated that three of the five exposed acidic residues around Trp171 in P. chrysosporium LiP affected the kinetics of VA oxidation (Smith and Doyle, 2006) and suggested that a partially acidic environment would stabilize the VA cation radical (VA•+) formed to act as an enzyme-bond mediator (Khindaria et al., 1997). Two of the acidic residues in LiP (Glu168 and Glu250) are conserved in VP, and the three others were introduced as single and/or multiple VP mutations. In contrast with the initial expectations that predicted a more efficient oxidation of VA, the N256D/R257D/A260F VP variant completely lost its activity on VA. Moreover, this and other variants bearing the R257D, N256D or S158E mutations also lost their activity on RB5. Interestingly, the RB5-oxidizing activity of the R257D variant was restored by adding VA to the reaction mixture. This suggested stabilization of VA•+ by a more acidic Trp164 environment in the mutated VP, similar to that found in LiP. Such behaviour is similar to the VA-mediated activity of LiP on RB5 and other substrates, and contrasted with RB5 direct oxidation by native VP (Heinfling et al., 1998b). Finally, transient-state kinetic studies revealed that combination of the A260F and R257A mutations in a VP double variant strongly increased the apparent second-order rate constants for the reduction of Compounds I and II by VA (over 20-fold and 50-fold improvement, respectively). Moreover, the first-order rate and dissociation constants for Compound II reduction showed improvement of both the k3 and KD3 constants. It is noteworthy that the k3app for reduction of Compound II of the R257A/A260F variant by VA (68 mM−1 s−1) was only slightly lower than reported for P. chrysosporium LiP (185 mM−1 s−1); and its k3 (46 s−1) was even higher than that of LiP (34 s−1) (Gelpke et al., 2002). The involvement of Phe267 in VA binding had been reported in LiP (Gelpke et al., 2002). The VP A260F and R257L single variants also showed some decreases of VA KD3, but the k3 values were not comparable to that of the VP double variant, indicating that the simultaneous removal of Arg257 and incorporation of a phenylalanine residue (homologous to LiP Phe267) are required to obtain a VP variant with VA transient-state kinetic constants comparable to those of LiP. It has been suggested that the discrepancy between the strongly improved transient-state constants and the barely modified steady-state constants of the R257/A260F variant of VP could be due to slow dissociation of the enzyme–product complex (Ruiz-Dueñas et al., 2008a). However, if VA•+ is the product of VA oxidation by VP and LiP, and veratraldehyde is formed by subsequent reaction with O2 (Schoemaker et al., 1994b), the latter reaction could also be the rate-limiting step in veratraldehyde formation by this variant. Oxidation of low redox-potential substrates at the VP main haem channel Sigmoidal curves are obtained for oxidation of different concentrations of low redox-potential 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS), p-hydroquinone, and 2,6-dimethoxyphenol by VP (Heinfling et al., 1998b; Ruiz-Dueñas et al., 2001) enabling the calculation of two sets of Km and kcat constants. This double kinetics suggested two independent oxidation sites, characterized by high and low specificity constants. This was confirmed by site-directed mutagenesis of VP Trp164 resulting in the removal of the high-specificity site for ABTS oxidation, whereas the low-specificity site remained (Ruiz-Dueñas et al., 2008a). Recently, a similar effect of the W164S mutation has been found on the VP oxidation of phenols. Simultaneously, the location of the low specificity oxidation site has been identified by site-directed mutagenesis at the main haem access channel. This resulted in several-fold increased specificity constants for phenols after removing the side-chains of some amino acids increasing the channel size (Ruiz-Dueñas et al., 2008b). To confirm substrate oxidation at the haem channel in these improved variants, the mutations opening the haem channel were combined with the W164S mutation. Variants with specificity constants similar to those of the high specificity site in native VP were obtained in spite of the removal of Trp164. These studies provided evidence on the oxidation of different substrates at the main haem channel of native VP, albeit with a lower specificity than observed at the catalytic Trp164 (that, in addition, is able to oxidize high redox-potential substrates that are not oxidized at the haem channel of native VP). This third substrate-oxidation site in VP is similar to the classical oxidation site reported in different peroxidases, including CIP (Smith et al., 1998; Tsukamoto et al., 1999). Interestingly, its operation is improved when the haem channel in VP, which is smaller than in CIP and plant peroxidases, is opened by site-directed mutagenesis. Studies currently being conducted include transient-state kinetics and crystallographic studies of these haem channel variants. It is possible to conclude that the versatile catalytic properties of P. eryngii VP are due to a hybrid molecular architecture that includes in the same protein different oxidation sites (for Mn2+, high redox-potential aromatics, and low redox-potential phenols and dyes) related to those found in the other basidiomycete peroxidase families (MnP, LiP, and CIP, respectively). Biotechnological interest of VP and other basidiomycete peroxidases Xylanases were introduced in the bleaching plant of paper-pulp mills, as a substitute for chlorine-containing reagents (Viikari et al., 1994), at a time when barely any information on ligninolytic peroxidases was available. However, enzymes acting on lignin (ligninolytic peroxidases and laccases) are the biocatalysts of choice for environmentally sound bleaching (Bajpai, 2004; Sigoillot et al., 2005). Laccases have been known for decades and are already produced in high yields, however, ligninolytic peroxidases are not commercially available despite the considerable efforts devoted to improve their heterologous expression (Conesa et al., 2000, 2002; Gu et al., 2003; Lú-Chau et al., 2004; Eibes et al., 2008). Laccases have low redox-potential and can degrade lignin only in the presence of mediators, whose costs and potential environmental risks are the main obstacles for implementing the laccase-mediator systems at the mill scale. By contrast, some ligninolytic peroxidases do not require mediators to degrade high redox-potential compounds. Among the different basidiomycete peroxidases, VP presents particular interest due to its catalytic versatility including the degradation of compounds that other peroxidases are not able to oxidize directly. VP versatility permits its application in Mn3+-mediated or Mn-independent reactions on both low and high redox-potential aromatic substrates and dyes, among others. It has been shown recently that VP can be used to reoxidize Mn-containing polyoxometalates, which are efficient oxidizers in paper pulp delignification (Marques et al., 2008). Another aspect of VP catalysis, which has been reported recently and merits investigation in more detail for some applications, is its high activity in the presence of organic solvents (Rodakiewicz-Nowak et al., 2006). The ability to degrade directly a variety of recalcitrant compounds represents a VP characteristic of the highest biotechnological interest. The use of aromatic mediators acting via their cation radicals (Schoemaker et al., 1994b) is required for LiP oxidation of simple phenols (Koduri and Tien, 1995), monomethoxylated aromatics (Harvey et al., 1986), polymeric dyes including Poly R-478 (Harvey et al., 1995; Paszczynski and Crawford, 1991), PAH (Barr and Aust, 1994), high redox-potential dyes (Heinfling et al., 1998b), ferrocytochrome c (Sheng and Gold., 1998), and, most important, polymeric lignin (Hammel et al., 1993). By contrast, VP does not requires mediators to oxidize many of the above compounds, including phenolic and non-phenolic aromatic pollutants (Rodríguez et al., 2004), pesticides (Dávila-Vázquez et al., 2005), high redox-potential and polymeric dyes (Heinfling et al., 1998a; Kamitsuji et al., 2005; Tinoco et al., 2007), PAH (Wang et al., 2003), and lignin (Camarero et al., 2001; Moreira et al., 2007). This VP ability represents an important advantage with respect to LiP, since the cost of VA as a mediator can be saved. Moreover, for more efficient oxidation of some recalcitrant compounds VP can also be used in combination with redox mediators including natural phenolic compounds (Tinoco et al., 2007). The abundant information on peroxidase structure–function relationships currently available has already been used to modulate the catalytic and operational properties of these enzymes (including peroxide inactivation resistance) as industrial biocatalysts using site-directed mutagenesis (Yeung et al., 1997; Wilcox et al., 1998; Timofeevski et al., 1999; Reading and Aust, 2000; Celik et al., 2001; Mester and Tien, 2001b; Miyazaki and Takahashi, 2001; Valderrama et al., 2002; Feng et al., 2003; Ryan et al., 2006; Pfister et al., 2007; Ruiz-Dueñas et al., 2008b). In those cases where the structural basis of the property to be improved is unknown, or too difficult to be predicted, directed evolution and/or combinatorial mutagenesis are the approach of choice (Cherry et al., 1999; Miyazaki-Imamura et al., 2003; Ryu et al., 2008). It is expected that genetic engineering will also contribute to overcoming the main drawback for the industrial utilization of VP and other ligninolytic peroxidases, i.e. their low yields in heterologous expression. An alternative approach is to engineer already commercial (i.e. high-yield expressed) peroxidases by introducing the ability to oxidize high-redox potential compounds by site-directed mutagenesis, based on the structure–function information already available. Using a combination of site-directed mutagenesis, saturation mutagenesis, directed evolution, and other molecular biology techniques, tailor-made biocatalysts will be available in the future enabling industrial exploitation of the unique catalytic abilities of the high redox-potential peroxidases (VP, LiP, and MnP) produced by the lignin-degrading basidiomycetes. Abbreviations Abbreviations ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) CIP Coprinopsis cinerea peroxidase EPR electron paramagnetic resonance MnP manganese peroxidase LiP lignin peroxidase LRET long-range electron transfer PAH polycyclic aromatic hydrocarbons RB5 Reactive Black 5 VA veratryl alcohol VA•+ VA cation radical VP versatile peroxidase This work was supported by the Spanish project BIO2005-03569 and BIO2008-01533, and the BIORENEW EU-project (NMP2-CT-2006-026456). The authors thank Klaus Piontek (Albert-Ludwigs University of Freiburg, Germany), María J Mate (CIB, CSIC, Madrid), and Antonio Romero (CIB, CSIC, Madrid) for crystallographic data; Rebecca Pogni and Riccardo Basosi (University of Sienna, Italy) for EPR analyses; and Andrew T Smith (University of Sussex, Brighton, UK) for helpful comments. FJR-D, EG, and YM thank the EU for a project contract, and MM thanks CSIC for an I3P fellowship. References Ambert-Balay K,  Fuchs SM,  Tien M.  Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis,  Biochemistry and Biophysics Research Communications ,  1998, vol.  251 (pg.  283- 286) Google Scholar CrossRef Search ADS   Ayala-Aceves M,  Baratto MC,  Basosi R,  Vázquez-Duhalt R,  Pogni R.  Spectroscopic characterization of a manganese–lignin peroxidase hybrid isozyme produced by Bjerkandera adusta in the absence of manganese: evidence of a protein-centred radical by hydrogen peroxide,  Journal of Molecular Catalysis B: Enzymatic ,  2001, vol.  16 (pg.  159- 167) Google Scholar CrossRef Search ADS   Bajpai P.  Biological bleaching of chemical pulps,  Critical Reviews in Biotechnology ,  2004, vol.  24 (pg.  1- 58) Google Scholar CrossRef Search ADS PubMed  Barr DP,  Aust SD.  Mechanisms white rot fungi use to degrade pollutants,  Environmental Science and Technology ,  1994, vol.  28 (pg.  A78- A87) Google Scholar CrossRef Search ADS   Baunsgaard L,  Dalboge H,  Houen G,  Rasmussen EM,  Welinder KG.  Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase: a new family of fungal peroxidases,  European Journal of Biochemistry ,  1993, vol.  213 (pg.  605- 611) Google Scholar CrossRef Search ADS PubMed  Blodig W,  Doyle WA,  Smith AT,  Winterhalter K,  Choinowski TH,  Piontek K.  Autocatalytic formation of hydroxy group at Cβ of Trp171 in lignin peroxidase,  Biochemistry ,  1998, vol.  37 (pg.  8832- 8838) Google Scholar CrossRef Search ADS PubMed  Blodig W,  Smith AT,  Doyle WA,  Piontek K.  Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171. Implications for the reaction mechanism,  Journal of Molecular Biology ,  2001, vol.  305 (pg.  851- 861) Google Scholar CrossRef Search ADS PubMed  Blodig W,  Smith AT,  Winterhalter K,  Piontek K.  Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase,  Archives of Biochemistry and Biophysics ,  1999, vol.  370 (pg.  86- 92) Google Scholar CrossRef Search ADS PubMed  Bourbonnais R,  Paice MG.  Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation,  FEBS Letters ,  1990, vol.  267 (pg.  99- 102) Google Scholar CrossRef Search ADS PubMed  Camarero S,  Bocchini P,  Galletti GC,  Martínez MJ,  Martínez AT.  Compositional changes of wheat lignin by a fungal peroxidase analyzed by Pyrolysis-GC/MS,  Journal of Analytical and Applied Pyrolysis ,  2001, vol.  58/59 (pg.  413- 423) Google Scholar CrossRef Search ADS   Camarero S,  Ibarra D,  Martínez MJ,  Martínez AT.  Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes,  Applied and Environmental Microbiology ,  2005, vol.  71 (pg.  1775- 1784) Google Scholar CrossRef Search ADS PubMed  Camarero S,  Sarkar S,  Ruiz-Dueñas FJ,  Martínez MJ,  Martínez AT.  Description of a versatile peroxidase involved in natural degradation of lignin that has both Mn-peroxidase and lignin-peroxidase substrate binding sites,  Journal of Biological Chemistry ,  1999, vol.  274 (pg.  10324- 10330) Google Scholar CrossRef Search ADS PubMed  Celik A,  Cullis PM,  Sutcliffe MJ,  Sangar R,  Raven EL.  Engineering the active site of ascorbate peroxidase,  European Journal of Biochemistry ,  2001, vol.  268 (pg.  78- 85) Google Scholar CrossRef Search ADS PubMed  Cherry JR,  Lamsa MH,  Schneider P,  Vind J,  Svendsen A,  Jones A,  Pedersen AH.  Directed evolution of a fungal peroxidase,  Nature Biotechnology ,  1999, vol.  17 (pg.  379- 384) Google Scholar CrossRef Search ADS PubMed  Conesa A,  Jeenes D,  Archer DB,  vandenHondel CAMJ,  Punt PJ.  Calnexin overexpression increases manganese peroxidase production in Aspergillus niger,  Applied and Environmental Microbiology ,  2002, vol.  68 (pg.  846- 851) Google Scholar CrossRef Search ADS PubMed  Conesa A,  van den Hondel CAMJJ,  Punt PJ.  Studies on the production of fungal peroxidases in Aspergillus niger,  Applied and Environmental Microbiology ,  2000, vol.  66 (pg.  3016- 3023) Google Scholar CrossRef Search ADS PubMed  Cullen D,  Kersten PJ.  Brambl R,  Marzluf GA.  Enzymology and molecular biology of lignin degradation,  Mycota III. Biochemistry and molecular biology ,  2004 Berlin Springer(pg.  249- 273) Dávila-Vázquez G,  Tinoco R,  Pickard MA,  Vázquez-Duhalt R.  Transformation of halogenated pesticides by versatile peroxidase from Bjerkandera adusta,  Enzyme and Microbial Technology ,  2005, vol.  36 (pg.  223- 231) Google Scholar CrossRef Search ADS   Doyle WA,  Blodig W,  Veitch NC,  Piontek K,  Smith AT.  Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis,  Biochemistry ,  1998, vol.  37 (pg.  15097- 15105) Google Scholar CrossRef Search ADS PubMed  Du P,  Collins JR,  Loew GH.  Homology modeling of a haem protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase,  Protein Engineering ,  1992, vol.  5 (pg.  679- 691) Google Scholar CrossRef Search ADS PubMed  Eibes GM,  Lú-Chau TA,  Ruiz-Dueñas FJ,  Feijoo G,  Martínez MJ,  Martínez AT,  Lema JM.  Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts,  Bioprocesses and Biosystems Engineering ,  2008  DOI 10.1007/s00449-008-0231-7 English AM,  Tsaprailis G.  Catalytic and structure-function relationships in haem proteins,  Advances in Inorganic Chemistry ,  1995, vol.  43 (pg.  79- 125) Feng ML,  Tachikawa H,  Wang XT,  Pfister TD,  Gengenbach AJ,  Lu Y.  Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase,  Journal of Biological Inorganic Chemistry ,  2003, vol.  8 (pg.  699- 706) Google Scholar CrossRef Search ADS PubMed  Gelpke MDS,  Lee J,  Gold MH.  Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L,  Biochemistry ,  2002, vol.  41 (pg.  3498- 3506) Google Scholar CrossRef Search ADS PubMed  Gelpke MDS,  Youngs HL,  Gold MH.  Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants,  European Journal of Biochemistry ,  2000, vol.  267 (pg.  7038- 7045) Google Scholar CrossRef Search ADS PubMed  Gerini MF,  Roccatano D,  Baciocchi E,  Di Nola A.  Molecular dynamics simulations of lignin peroxidase in solution,  Biophysical Journal ,  2003, vol.  84 (pg.  3883- 3893) Google Scholar CrossRef Search ADS PubMed  Giardina P,  Palmieri G,  Fontanella B,  Rivieccio V,  Sannia G.  Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust,  Archives of Biochemistry and Biophysics ,  2000, vol.  376 (pg.  171- 179) Google Scholar CrossRef Search ADS PubMed  Gold MH,  Youngs HL,  Gelpke MD.  Manganese peroxidase,  Metal Ions in Biological Systems ,  2000, vol.  37 (pg.  559- 586) Google Scholar PubMed  Gu L,  Lajoie C,  Kelly C.  Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris,  Biotechnology Progress ,  2003, vol.  19 (pg.  1403- 1409) Google Scholar CrossRef Search ADS PubMed  Guillén F,  Evans CS.  Anisaldehyde and veratraldehyde acting as redox cycling agents for H2O2 production by Pleurotus eryngii,  Applied and Environmental Microbiology ,  1994, vol.  60 (pg.  2811- 2817) Google Scholar PubMed  Hammel KE,  Cullen D.  Role of fungal peroxidases in biological ligninolysis,  Current Opinion in Plant Biology ,  2008, vol.  11 (pg.  349- 355) Google Scholar CrossRef Search ADS PubMed  Hammel KE,  Jensen KA,  Mozuch MD,  Landucci LL,  Tien M,  Pease EA.  Ligninolysis by a purified lignin peroxidase,  Journal of Biological Chemistry ,  1993, vol.  268 (pg.  12274- 12281) Google Scholar PubMed  Harvey PJ,  Candeias LP,  King PJ,  Palmer JM,  Wever R.  Lignin peroxidase catalysis: Reaction with veratryl alcohol and a polymeric dye, Poly R,  Biochemistry Society Transactions ,  1995, vol.  23 pg.  S340  Google Scholar CrossRef Search ADS   Harvey PJ,  Schoemaker HE,  Palmer JM.  Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium,  FEBS Letters ,  1986, vol.  195 (pg.  242- 246) Google Scholar CrossRef Search ADS   Heinfling A,  Martínez MJ,  Martínez AT,  Bergbauer M,  Szewzyk U.  Transformation of industrial dyes by manganese peroxidase from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction,  Applied and Environmental Microbiology ,  1998, vol.  64 (pg.  2788- 2793) Google Scholar PubMed  Heinfling A,  Ruiz-Dueñas FJ,  Martínez MJ,  Bergbauer M,  Szewzyk U,  Martínez AT.  A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta,  FEBS Letters ,  1998, vol.  428 (pg.  141- 146) Google Scholar CrossRef Search ADS PubMed  Higuchi T.  Microbial degradation of lignin: role of lignin peroxidase, manganese peroxidase, and laccase,  Proceedings of the Japanese Academy B ,  2004, vol.  80 (pg.  204- 214) Google Scholar CrossRef Search ADS   Hildén K,  Martínez AT,  Hatakka A,  Lundell T.  The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent,  Fungal Genetics and Biology ,  2005, vol.  42 (pg.  403- 419) Google Scholar CrossRef Search ADS PubMed  Hofrichter M,  Ullrich R.  Haem-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance,  Applied Microbiology and Biotechnology ,  2006, vol.  71 (pg.  276- 288) Google Scholar CrossRef Search ADS PubMed  Johjima T,  Itoh H,  Kabuto M,  Tokimura F,  Nakagawa T,  Wariishi H,  Tanaka H.  Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium,  Proceedings of the National Academy of Sciences, USA ,  1999, vol.  96 (pg.  1989- 1994) Google Scholar CrossRef Search ADS   Kamitsuji H,  Watanabe T,  Honda Y,  Kuwahara M.  Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isozyme from Pleurotus ostreatus without redox mediators,  Biochemical Journal ,  2005, vol.  386 (pg.  387- 393) Google Scholar CrossRef Search ADS PubMed  Kapich AN,  Steffen KT,  Hofrichter M,  Hatakka A.  Involvement of lipid peroxidation in the degradation of a non-phenolic lignin model compound by manganese peroxidase of the litter-decomposing fungus Stropharia coronilla,  Biochemical and Biophysical Research Communications ,  2005, vol.  330 (pg.  371- 377) Google Scholar CrossRef Search ADS PubMed  Kersten P,  Cullen D.  Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium,  Fungal Genetics and Biology ,  2007, vol.  44 (pg.  77- 87) Google Scholar CrossRef Search ADS PubMed  Kersten PJ,  Kirk TK.  Involvement of a new enzyme, glyoxal oxidase, in extracellular H2O2 production by Phanerochaete chrysosporium,  Journal of Bacteriology ,  1987, vol.  169 (pg.  2195- 2201) Google Scholar PubMed  Khindaria A,  Aust SD.  EPR detection and characterization of lignin peroxidase porphyrin π-cation radical,  Biochemistry ,  1996, vol.  35 (pg.  13107- 13111) Google Scholar CrossRef Search ADS PubMed  Khindaria A,  Nie G,  Aust SD.  Detection and characterization of the lignin peroxidase compound II–veratryl alcohol cation radical complex,  Biochemistry ,  1997, vol.  36 (pg.  14181- 14185) Google Scholar CrossRef Search ADS PubMed  Kirk TK,  Farrell RL.  Enzymatic ‘combustion’: the microbial degradation of lignin,  Annual Review of Microbiology ,  1987, vol.  41 (pg.  465- 505) Google Scholar CrossRef Search ADS PubMed  Kishi K,  Kusters-van Someren M,  Mayfield MB,  Sun J,  Loehr TM,  Gold MH.  Characterization of manganese(II) binding site mutants of manganese peroxidase,  Biochemistry ,  1996, vol.  35 (pg.  8986- 8994) Google Scholar CrossRef Search ADS PubMed  Koduri RS,  Tien M.  Oxidation of guaiacol by lignin peroxidase. Role of veratryl alcohol,  Journal of Biological Chemistry ,  1995, vol.  270 (pg.  22254- 22258) Google Scholar CrossRef Search ADS PubMed  Kusters-van Someren M,  Kishi K,  Lundell T,  Gold MH.  The manganese binding site of manganese peroxidase: characterization of an Asp179Asn site-directed mutant protein,  Biochemistry ,  1995, vol.  34 (pg.  10620- 10627) Google Scholar CrossRef Search ADS PubMed  Larrondo L,  Gonzalez A,  Perez AT,  Cullen D,  Vicuna R.  The nop gene from Phanerochaete chrysosporium encodes a peroxidase with novel structural features,  Biophysical Chemistry ,  2005, vol.  116 (pg.  167- 173) Google Scholar CrossRef Search ADS PubMed  Lisov AV,  Leontievsky AA,  Golovleva LA.  Hybrid Mn-peroxidases from basidiomycetes,  Applied Biochemistry and Microbiology ,  2007, vol.  43 (pg.  536- 543) Google Scholar CrossRef Search ADS   Lú-Chau TA,  Ruiz-Dueñas FJ,  Camarero S,  Feijoo G,  Martínez MJ,  Lema JM,  Martínez AT.  Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans,  Bioprocesses and Biosystems Engineering ,  2004, vol.  26 (pg.  287- 293) Google Scholar CrossRef Search ADS   Maeda Y,  Kajiwara S,  Ohtaguchi K.  Manganese peroxidase gene of the perennial mushroom Elfvingia applanata: cloning and evaluation of its relationship with lignin degradation,  Biotechnology Letters ,  2001, vol.  23 (pg.  103- 109) Google Scholar CrossRef Search ADS   Magliozzo RS,  Marcinkeviciene JA.  The role of Mn(II)-peroxidase activity of mycobacterial catalase-peroxidase in activation of the antibiotic isoniazid,  Journal of Biological Chemistry ,  1997, vol.  272 (pg.  8867- 8870) Google Scholar CrossRef Search ADS PubMed  Marques G,  Gamelas JAF,  Evtuguin D,  Ruiz-Dueñas FJ,  Morales M,  del Río JC,  Martínez AT,  Gutiérrez A.  Versatile peroxidase reoxidation of Mn-based polyoxomethalate in chlorine-free bleaching of eucalypt pulp,  Proceedings of the 8th international peroxidase symposium. Tampere ,  2008  20–23 August Martínez AT.  Molecular biology and structure-function of lignin-degrading haem peroxidases,  Enzyme and Microbial Technology ,  2002, vol.  30 (pg.  425- 444) Google Scholar CrossRef Search ADS   Martínez AT.  Polaina J,  MacCabe AP.  High redox potential peroxidases,  Industrial enzymes: structure, function and applications ,  2007 Berlin Springer(pg.  475- 486) Martínez AT,  Speranza M,  Ruiz-Dueñas FJ,  Ferreira P,  Camarero S,  Guillén F,  Martínez MJ,  Gutiérrez A,  del Río JC.  Biodegradation of lignocellulosics: microbiological, chemical and enzymatic aspects of fungal attack to lignin,  International Microbiology ,  2005, vol.  8 (pg.  195- 204) Google Scholar PubMed  Martínez MJ,  Ruiz-Dueñas FJ,  Guillén F,  Martínez AT.  Purification and catalytic properties of two manganese-peroxidase isoenzymes from Pleurotus eryngii,  European Journal of Biochemistry ,  1996, vol.  237 (pg.  424- 432) Google Scholar CrossRef Search ADS PubMed  Matsubara M,  Suzuki J,  Deguchi T,  Miura M,  Kitaoka Y.  Characterization of manganese peroxidases from the hyperligninolytic fungus IZU-154,  Applied and Environmental Microbiology ,  1996, vol.  62 (pg.  4066- 4072) Google Scholar PubMed  Mester T,  Ambert-Balay K,  Ciofi-Baffoni S,  Banci L,  Jones AD,  Tien M.  Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase,  Journal of Biological Chemistry ,  2001, vol.  276 (pg.  22985- 22990) Google Scholar CrossRef Search ADS PubMed  Mester T,  Tien M.  Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium,  Biochemical and Biophysical Research Communications ,  2001, vol.  284 (pg.  723- 728) Google Scholar CrossRef Search ADS PubMed  Miki Y,  Tanaka H,  Nakamura M,  Wariishi H.  Isolation and characterization of a novel lignin peroxidase from the white-rot basidiomycete Trametes cervina,  Journal of the Faculty of Agriculture of Kyushu University ,  2006, vol.  51 (pg.  99- 104) Miyazaki C,  Takahashi H.  Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2,  FEBS Letters ,  2001, vol.  509 (pg.  111- 114) Google Scholar CrossRef Search ADS PubMed  Miyazaki-Imamura C,  Oohira K,  Kitagawa R,  Nakano H,  Yamane T,  Takahashi H.  Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase,  Protein Engineering ,  2003, vol.  16 (pg.  423- 428) Google Scholar CrossRef Search ADS PubMed  Moreira PR,  Almeida-Vara E,  Malcata FX,  Duarte JC.  Lignin transformation by a versatile peroxidase from a novel Bjerkandera sp strain,  International Biodeterioration and Biodegradation ,  2007, vol.  59 (pg.  234- 238) Google Scholar CrossRef Search ADS   Moreira PR,  Duez C,  Dehareng D,  Antunes A,  Almeida-Vara E,  Frère JM,  Malcata FX,  Duarte JC.  Molecular characterisation of a versatile peroxidase from a Bjerkandera strain,  Journal of Biotechnology ,  2005, vol.  118 (pg.  339- 352) Google Scholar CrossRef Search ADS PubMed  Morgenstern I,  Klopman S,  Hibbett DS.  Molecular evolution and diversity of lignin degrading haem peroxidases in the agaricomycetes,  Journal of Molecular Evolution ,  2008, vol.  66 (pg.  243- 257) Google Scholar CrossRef Search ADS PubMed  Paszczynski A,  Crawford RL.  Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol,  Biochemistry and Biophysics Research Communications ,  1991, vol.  178 (pg.  1056- 1063) Google Scholar CrossRef Search ADS   Pelletier H,  Kraut J.  Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c,  Science ,  1992, vol.  258 (pg.  1748- 1755) Google Scholar CrossRef Search ADS PubMed  Pérez-Boada M,  Doyle WA,  Ruiz-Dueñas FJ,  Martínez MJ,  Martínez AT,  Smith AT.  Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimisation of in vitro folding,  Enzyme and Microbial Technology ,  2002, vol.  30 (pg.  518- 524) Google Scholar CrossRef Search ADS   Pérez-Boada M,  Ruiz-Dueñas FJ,  Pogni R,  Basosi R,  Choinowski T,  Martínez MJ,  Piontek K,  Martínez AT.  Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigations of three long-range electron transfer pathways,  Journal of Molecular Biology ,  2005, vol.  354 (pg.  385- 402) Google Scholar CrossRef Search ADS PubMed  Pfister TD,  Mirarefi AY,  Gengenbach AJ, et al.  Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase,  Journal of Biological Inorganic Chemistry ,  2007, vol.  12 (pg.  126- 137) Google Scholar CrossRef Search ADS PubMed  Pogni R,  Baratto MC,  Giansanti S,  Teutloff C,  Verdin J,  Valderrama B,  Lendzian F,  Lubitz W,  Vázquez-Duhalt R,  Basosi R.  Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta,  Biochemistry ,  2005, vol.  44 (pg.  4267- 4274) Google Scholar CrossRef Search ADS PubMed  Pogni R,  Baratto MC,  Teutloff C,  Giansanti S,  Ruiz-Dueñas FJ,  Choinowski T,  Piontek K,  Martínez AT,  Lendzian F,  Basosi R.  A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multi-frequency EPR and DFT study,  Journal of Biological Chemistry ,  2006, vol.  281 (pg.  9517- 9526) Google Scholar CrossRef Search ADS PubMed  Pogni R,  Teutloff C,  Lendzian F,  Basosi R.  Tryptophan radicals as reaction intermediates in versatile peroxidases: multifrequency EPR, ENDOR and density functional theory studies,  Applied Magnetic Resonance ,  2007, vol.  31 (pg.  509- 526) Google Scholar CrossRef Search ADS   Reading NS,  Aust SD.  Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability,  Biotechnology Progress ,  2000, vol.  16 (pg.  326- 333) Google Scholar CrossRef Search ADS PubMed  Rodakiewicz-Nowak J,  Jarosz-Wilkolazka A,  Luterek J.  Catalytic activity of versatile peroxidase from Bjerkandera fumosa in aqueous solutions of water-miscible organic solvents,  Applied Catalysis A: General ,  2006, vol.  308 (pg.  56- 61) Google Scholar CrossRef Search ADS   Rodríguez E,  Nuero O,  Guillén F,  Martínez AT,  Martínez MJ.  Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase,  Soil Biology and Biochemistry ,  2004, vol.  36 (pg.  909- 916) Google Scholar CrossRef Search ADS   Ruiz-Dueñas FJ,  Aguilar A,  Martínez MJ,  Zorn H,  Martínez AT.  Gene cloning, heterologous expression, in vitro reconstitution, and catalytic properties of a versatile peroxidase,  Biocatalysis and Biotransformation ,  2007, vol.  25 (pg.  276- 285) Google Scholar CrossRef Search ADS   Ruiz-Dueñas FJ,  Camarero S,  Pérez-Boada M,  Martínez MJ,  Martínez AT.  A new versatile peroxidase from Pleurotus,  Biochemistry Society Transactions ,  2001, vol.  29 (pg.  116- 122) Google Scholar CrossRef Search ADS   Ruiz-Dueñas FJ,  Martínez MJ,  Martínez AT.  Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii,  Molecular Microbiology ,  1999, vol.  31 (pg.  223- 236) Google Scholar CrossRef Search ADS PubMed  Ruiz-Dueñas FJ,  Morales M,  Mate MJ,  Romero A,  Martínez MJ,  Smith AT,  Martínez AT.  Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase,  Biochemistry ,  2008, vol.  47 (pg.  1685- 1695) Google Scholar CrossRef Search ADS PubMed  Ruiz-Dueñas FJ,  Morales M,  Pérez-Boada M,  Choinowski T,  Martínez MJ,  Piontek K,  Martínez AT.  Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic and crystallographic study,  Biochemistry ,  2007, vol.  46 (pg.  66- 77) Google Scholar CrossRef Search ADS PubMed  Ruiz-Dueñas FJ,  Morales M,  Rencoret J,  Gutiérrez A,  del Río JC,  Martínez MJ,  Martínez AT. ,  Improved peroxidases. Patent (Spain) P200801292 ,  2008 Ruiz-Dueñas FJ,  Pogni R,  Morales M,  Martínez MJ,  Basosi R,  Martínez AT.  Tailoring alternative catalytic protein radicals in Pleurotus eryngii versatile peroxidase,  Proceedings of the 8th International Peroxidase Symposium. Tampere ,  2008  20–23 August Ryan BJ,  Carolan N,  O'Fagain C.  Horseradish and soybean peroxidases: comparable tools for alternative niches?,  Trends in Biotechnology ,  2006, vol.  24 (pg.  355- 363) Google Scholar CrossRef Search ADS PubMed  Ryu K,  Hwang SY,  Kim KH,  Kang JH,  Lee EK.  Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability,  Journal of Biotechnology ,  2008, vol.  133 (pg.  110- 115) Google Scholar CrossRef Search ADS PubMed  Schoemaker HE,  Lundell TK,  Floris R,  Glumoff T,  Winterhalter KH,  Piontek K.  Do carbohydrates play a role in lignin peroxidase cycle? Redox catalysis in the endergonic region of the driving force,  Bioorganic and Medicinal Chemistry ,  1994, vol.  2 (pg.  509- 519) Google Scholar CrossRef Search ADS PubMed  Schoemaker HE,  Lundell TK,  Hatakka AI,  Piontek K.  The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: the redox cycle revisited,  FEMS Microbiology Reviews ,  1994, vol.  13 (pg.  321- 332) Google Scholar CrossRef Search ADS   Sharp KH,  Mewies M,  Moody PC,  Raven EL.  Crystal structure of the ascorbate peroxidase–ascorbate complex,  Nature Structural Biology ,  2003, vol.  10 (pg.  303- 307) Google Scholar CrossRef Search ADS PubMed  Sheng D,  Gold MH.  Irreversible oxidation of ferricytochrome c by lignin peroxidase,  Biochemistry ,  1998, vol.  37 (pg.  2029- 2036) Google Scholar CrossRef Search ADS PubMed  Sigoillot C,  Camarero S,  Vidal T, et al.  Comparison of different fungal enzymes for bleaching high-quality paper pulps,  Journal of Biotechnology ,  2005, vol.  115 (pg.  333- 343) Google Scholar CrossRef Search ADS PubMed  Smith AT,  Doyle WA.  Engineered peroxidases with veratryl alcohol oxidase activity,  Patent (International) WO/2006–114616 ,  2006 Smith AT,  Veitch NC.  Substrate binding and catalysis in haem peroxidases,  Current Opinion in Chemical Biology ,  1998, vol.  2 (pg.  269- 278) Google Scholar CrossRef Search ADS PubMed  Sundaramoorthy M,  Kishi K,  Gold MH,  Poulos TL.  The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06 Å resolution,  Journal of Biological Chemistry ,  1994, vol.  269 (pg.  32759- 32767) Google Scholar PubMed  Sundaramoorthy M,  Kishi K,  Gold MH,  Poulos TL.  Crystal structures of substrate binding site mutants of manganese peroxidase,  Journal of Biological Chemistry ,  1997, vol.  272 (pg.  17574- 17580) Google Scholar CrossRef Search ADS PubMed  Sundaramoorthy M,  Youngs HL,  Gold MH,  Poulos TL.  High-resolution crystal structure of manganese peroxidase: substrate and inhibitor complexes,  Biochemistry ,  2005, vol.  44 (pg.  6463- 6470) Google Scholar CrossRef Search ADS PubMed  Tamura K,  Dudley J,  Nei M,  Kumar S.  MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0,  Molecular Biology and Evolution ,  2007, vol.  24 (pg.  1596- 1599) Google Scholar CrossRef Search ADS PubMed  Timofeevski SL,  Nie G,  Reading NS,  Aust SD.  Engineering a functional hybrid of manganese peroxidase and lignin peroxidase,  Plant Peroxidase Newsletter ,  1999, vol.  13 (pg.  99- 111) Tinoco R,  Verdín J,  Vázquez-Duhalt R.  Role of oxidizing mediators and tryptophan 172 in the decoloration of industrial dyes by the versatile peroxidase from Bjerkandera adusta,  Journal of Molecular Catalysis B: Enzymatic ,  2007, vol.  46 (pg.  1- 7) Google Scholar CrossRef Search ADS   Tsukamoto K,  Itakura H,  Sato K,  Fukuyama K,  Miura S,  Takahashi S,  Ikezawa H,  Hosoya T.  Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics,  Biochemistry ,  1999, vol.  38 (pg.  12558- 12568) Google Scholar CrossRef Search ADS PubMed  Tsukihara T,  Honda Y,  Sakai R,  Watanabe T,  Watanabe T.  Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2,  Applied and Environmental Microbiology ,  2008, vol.  74 (pg.  2873- 2881) Google Scholar CrossRef Search ADS PubMed  Valderrama B,  Ayala M,  Vázquez-Duhalt R.  Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes,  Chemistry and Biology ,  2002, vol.  9 (pg.  555- 565) Google Scholar CrossRef Search ADS PubMed  Veitch NC.  Horseradish peroxidase: a modern view of a classic enzyme,  Phytochemistry ,  2004, vol.  65 (pg.  249- 259) Google Scholar CrossRef Search ADS PubMed  Viikari L,  Kantelinen A,  Sundquist J,  Linko M.  Xylanases in bleaching: from an idea to the industry,  FEMS Microbiology Reviews ,  1994, vol.  13 (pg.  335- 350) Google Scholar CrossRef Search ADS   Wang YX,  Vázquez-Duhalt R,  Pickard MA.  Manganese–lignin peroxidase hybrid from Bjerkandera adusta oxidizes polycyclic aromatic hydrocarbons more actively in the absence of manganese,  Canadian Journal of Microbiology ,  2003, vol.  49 (pg.  675- 682) Google Scholar CrossRef Search ADS PubMed  Welinder KG.  Superfamily of plant, fungal and bacterial peroxidases,  Current Opinion in Structural Biology ,  1992, vol.  2 (pg.  388- 393) Google Scholar CrossRef Search ADS   Whitwam RE,  Brown KR,  Musick M,  Natan MJ,  Tien M.  Mutagenesis of the Mn2+-binding site of manganese peroxidase affects oxidation of Mn2+ by both compound I and compound II.,  Biochemistry ,  1997, vol.  36 (pg.  9766- 9773) Google Scholar CrossRef Search ADS PubMed  Wilcox SK,  Putnam CD,  Sastry M,  Blankenship J,  Chazin WJ,  McRee DE,  Goodin DB.  Rational design of a functional metalloenzyme: Introduction of a site for manganese binding and oxidation into a haem peroxidase,  Biochemistry ,  1998, vol.  37 (pg.  16853- 16862) Google Scholar CrossRef Search ADS PubMed  Yeung BKS,  Wang XT,  Sigman JA,  Petillo PA,  Lu Y.  Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase,  Chemistry and Biology ,  1997, vol.  4 (pg.  215- 221) Google Scholar CrossRef Search ADS PubMed  Youngs HL,  Gelpke MDS,  Li DM,  Sundaramoorthy M,  Gold MH.  The role of Glu39 in Mn-II binding and oxidation by manganese peroxidase from Phanerochaete chrysosporium,  Biochemistry ,  2001, vol.  40 (pg.  2243- 2250) Google Scholar CrossRef Search ADS PubMed  © The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Experimental Botany Oxford University Press

Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases

Download PDF
12 pages

 
/lp/oxford-university-press/substrate-oxidation-sites-in-versatile-peroxidase-and-other-nx1ZxB2pVh

References (113)

Publisher
Oxford University Press
Copyright
© The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org
ISSN
0022-0957
eISSN
1460-2431
DOI
10.1093/jxb/ern261
pmid
18987391
Publisher site
See Article on Publisher Site

Abstract

Abstract Versatile peroxidase (VP) is defined by its capabilities to oxidize the typical substrates of other basidiomycete peroxidases: (i) Mn2+, the manganese peroxidase (MnP) substrate (Mn3+ being able to oxidize phenols and initiate lipid peroxidation reactions); (ii) veratryl alcohol (VA), the typical lignin peroxidase (LiP) substrate; and (iii) simple phenols, which are the substrates of Coprinopsis cinerea peroxidase (CIP). Crystallographic, spectroscopic, directed mutagenesis, and kinetic studies showed that these ‘hybrid’ properties are due to the coexistence in a single protein of different catalytic sites reminiscent of those present in the other basidiomycete peroxidase families. Crystal structures of wild and recombinant VP, and kinetics of mutated variants, revealed certain differences in its Mn-oxidation site compared with MnP. These result in efficient Mn2+ oxidation in the presence of only two of the three acidic residues forming its binding site. On the other hand, a solvent-exposed tryptophan is the catalytically-active residue in VA oxidation, initiating an electron transfer pathway to haem (two other putative pathways were discarded by mutagenesis). Formation of a tryptophanyl radical after VP activation by peroxide was detected using electron paramagnetic resonance. This was the first time that a protein radical was directly demonstrated in a ligninolytic peroxidase. In contrast with LiP, the VP catalytic tryptophan is not β-hydroxylated under hydrogen peroxide excess. It was also shown that the tryptophan environment affected catalysis, its modification introducing some LiP properties in VP. Moreover, some phenols and dyes are oxidized by VP at the edge of the main haem access channel, as found in CIP. Finally, the biotechnological interest of VP is discussed. Crystal structures, fungal peroxidases, haem access-channel, lignin biodegradation, manganese-binding site, site-directed mutagenesis, spectroscopic analyses, transient-state kinetics, tryptophanyl radical, versatile peroxidase Ligninolytic peroxidases Degradation of lignin is a key process for carbon recycling in land ecosystems, as well as for utilization of lignocellulosic biomass in paper pulp manufacture and bioethanol production. Degradation of this recalcitrant aromatic polymer is caused in nature by wood-rotting fungi through a process that was defined as an enzymatic combustion (Kirk and Farrell, 1987). Several oxidoreductases (including peroxidases, oxidases, and laccases) secreted by ligninolytic basidiomycetes, the so-called white-rot fungi, are involved in lignin biodegradation (Higuchi, 2004; Martínez et al., 2005; Kersten and Cullen, 2007). Ligninolytic haem peroxidases are able to break down model compounds representative of the main linkage types in lignin due to their high redox-potential and specialized catalytic mechanisms (Martínez, 2002, 2007; Hammel and Cullen, 2008). Extracellular oxidases, such as glyoxal oxidase (a copper-radical protein) (Kersten and Kirk, 1987) and aryl-alcohol oxidase (a flavoprotein) (Guillén and Grans, 1994), provide the hydrogen peroxide required by peroxidases. Finally, laccases can directly oxidize only minor phenolic lignin moieties, although their biotechnological interest has increased after discovering that redox mediators (Bourbonnais and Paice, 1990), including natural phenols (Camarero et al., 2005), strongly enhance their oxidizing power. Recently, an extracellular haem-thiolate peroxidase, which is also able to degrade lignin model compounds, among other enzymatic reactions, has been found in soil Agaricales, such as Agrocybe aegerita (V Briganti) Singer and several Coprinus species (Hofrichter and Ullrich, 2006). Ligninolytic haem peroxidases from white-rot fungi include lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP) (Gold et al., 2000; Martínez, 2002; Hammel et al., 2008). Due to the high redox-potential required for lignin oxidation, ligninolytic peroxidases have been the object of high interest as industrial biocatalysts (Martínez, 2007). LiP and MnP were first described in Phanerochaete chrysosporium Burdsall (order Corticiales) and genes/cDNA encoding isoenzymes LiPH8 and MnP1 cloned in 1987–1988 from the same fungus (Cullen and Kersten, 2004). From this date, related genes have been cloned from other Corticiales, Agaricales, and Polyporales basidiomycetes including: (i) over 20 different mnp genes from Agaricus bisporus (J.E. Lange) Imbach, Ceriporiopsis rivulosa (Berk. & M.A. Curtis) Gilbertson & Ryvarden, Ceriporiopsis subvermispora (Pilat) Gilbertson & Ryvarden, Dichomitus squalens (P. Karsten) D.A. Reid, Lentinula edodes (Berk.) Pegler, Phlebia radiata Fries, P. chrysosporium, Phanerochaete sordida (P. Karsten) J. Eriksson & Ryvarden, Pleurotus ostreatus (Jacquin) P. Kummer, and Trametes versicolor (L.) Lloyd; and (ii) around 15 lip genes from Bjerkandera adusta (Willd.) P. Karst., P. radiata, P. chrysosporium, Trametes cervina (Schwein.) Bres., and T. versicolor (Ruiz-Dueñas et al., 2007a). By contrast, VP genes have been cloned only from a few species from the genera Pleurotus (order Agaricales), including Pleurotus eryngii (DC.) Gillet and Pleurotus sapidus (Schulzer) Sacc. among others, and Bjerkandera (order Polyporales), including Bjerkandera adusta (Willd.) P. Karst. (Camarero et al., 1999; Ruiz-Dueñas et al., 1999, 2007a; Moreira et al., 2005). VP could also be produced by other Polyporales basidiomycetes including species from the genera Panus (Lisov et al., 2007), Trametes (Ruiz-Dueñas et al., 2007a), and Spongipellis (GenBank AB244274). The evolutionary relationships of all the proteins encoded by these genes are shown in the dendrogram of Fig. 1, which also indicates the presence in the different sequences of residues forming the substrate oxidation sites discussed below (involved in the oxidation of Mn2+ and high redox-potential aromatic substrates). The dendrogram obtained from comparison of the 56 basidiomycete peroxidases available showed a well-defined cluster corresponding to typical MnP (from Phanerochaete, Ceriporiopsis, Phlebia, and Dichomitus). The second cluster is large and includes a small subcluster containing three atypical MnP from Ganoderma species (and the so-called Mn-repressed peroxidase from T. versicolor), and a large subcluster where all LiP and VP, and some MnP (from Pleurotus, Trametes, Phlebia, Lentinula, and Agaricus), are located (together with unclassified peroxidases, such as Trametes PGV and PGVII). In this heterogenous cluster, typical LiP (from Phanerochaete, Bjerkandera, Phlebia, and Trametes) form a relatively homogeneous group, whereas the group of typical VP (from Pleurotus) is small, and other VP are related to different MnP. Finally, two peroxidases from the non-lignicolous fungi Coprinopsis cinerea (Schaeff.) Redhead, Vilgalys and Moncalvo (synonym Coprinus cinereus (Schaeff.) Gray) and Coprinellus disseminatus (Pers.) Gray, and the NOPA peroxidase identified in the P. chrysosporium genome (Larrondo et al., 2005), are not grouped together suggesting that they are not related to ligninolytic peroxidases. Gene fragments from the above peroxidase families have been recently identified in a variety of other basidiomycetes indicating a wider distribution (Morgenstern et al., 2008). Fig. 1. View largeDownload slide Evolutionary relationships and structural–functional classification of basidiomycete peroxidases (mature proteins) including 53 ligninolytic enzymes belonging to the MnP, LiP, and VP families (and some related unclassified peroxidases) and three low-redox potential peroxidases. The phylogenetic analysis was performed using MEGA4 (Tamura et al., 2007) with Poisson-corrected evolutionary distances, complete deletion option (269 positions in the final dataset), and UPGMA clustering method. The structural–functional classification was based on the presence in the protein sequence or molecular structure of the structural motifs characteristic of the above families, namely: (i) Mn2+-oxidation site formed by three acidic residues (red circles); and (ii) exposed catalytic tryptophan oxidizing high redox-potential substrates (blue circles). As a result of the structural analysis, three putative LiP (previously described as MnP) and six putative VP (unclassified or described as LiP or MnP) were identified. The presence of a catalytic tyrosine in the LiP from T. cervina, playing the same function of the above catalytic tryptophan, is also indicated (blue square). Species abbreviations: AB, A. bisporus; B, Bjerkandera sp; BA, B. adusta; CC, C. cinerea; CD, C. disseminatus; CR, C. rivulosa; CS, C. subvermispora; DS, D. squalens; GA, G. applanatum; GAU, G. australe; GF, G. formosanum; IZU, unidentified basidiomycete IZU-154; LE, L. edodes; PC, P. chrysosporium; PE, P. eryngii; PO, P. ostreatus; PP, P. pulmonarius; PR, P. radiata; PS, P. sordida; PSA, P. sapidus; S, Spongipellis sp; TC, T. cervina; and TV, T. versicolor. Protein sequence entries: AB-MnP, CAG27835; B-VP, AAO47909; BA-LiP, 1906181A; CD-PerO, AAZ14938; CC-CIP, CAA50060; CR-MnPA, ABB83812; CR-MnPB, ABB83813; CS-MnP1, AAB03480; CS-MnP2, AAD43581; CS-MnP3, AAD45725; CS-MnP4, AAO61784; DS-MnP1, AAF31329; DS-MnP2, AAF31330; GA-MnP1, BAA88392; GAU-MnP, ABB77244; GF-MnP, ABB77243; IZU-MnP1, no entry available but sequence taken from the literature (Matsubara et al., 1996); LE-MnP1, BAE79199; PC-LiPA (isoenzyme H8), AAA53109; PC-LiPB (isoenzyme H8), AAA33741; PCLiPC (isoenzyme H10), AAA33739; PC-LiPD (isoenzyme H2), CAA33621; PC-LiPE (isoenzyme H8), AAA33738; PC-LiPF, AAA33736; PC-LiPH (isoenzyme H8), AAA56852; PC-LiPJ, AAD46494; PC-MnP1, AAA33744; PCMnP2, AAA33745; PC-MnP3, AAB39652; PC-NOPA, AAU82081; PE-VPL1, AAD01401; PE-VPL2, AAD01404; PE-VPL3, CAD56164 (and DQ056374); PE-VPS1, AAD54310; PO-MnP1, AAA84396; PO-MnP2, CAB51617; PO-MnP3, BAA33449; PP-MnP5, AAX40734; PR-LiP, P20010; PR-LiP1, AAW59419; PR-LiP4, AAW66483; PR-MnP2, CAC85963; PR-MnP3, CAC84573; PS-MnP1, BAC06185; PS-MnP2, BAC06186; PS-MnP3, BAC06187; PSA-VP, CAJ01576; S-MnP1, BAE79812; TC-LiP, AB191466; TV-LiP12, AAA34049; TV-LiP7, CAA83147; TV-LiPGII, CAA53333; TV-MnP2, CAA83148; TV-MRP, AAB63460; TV-PGV, CAA54398; and TV-PGVII, CAA91043. Fig. 1. View largeDownload slide Evolutionary relationships and structural–functional classification of basidiomycete peroxidases (mature proteins) including 53 ligninolytic enzymes belonging to the MnP, LiP, and VP families (and some related unclassified peroxidases) and three low-redox potential peroxidases. The phylogenetic analysis was performed using MEGA4 (Tamura et al., 2007) with Poisson-corrected evolutionary distances, complete deletion option (269 positions in the final dataset), and UPGMA clustering method. The structural–functional classification was based on the presence in the protein sequence or molecular structure of the structural motifs characteristic of the above families, namely: (i) Mn2+-oxidation site formed by three acidic residues (red circles); and (ii) exposed catalytic tryptophan oxidizing high redox-potential substrates (blue circles). As a result of the structural analysis, three putative LiP (previously described as MnP) and six putative VP (unclassified or described as LiP or MnP) were identified. The presence of a catalytic tyrosine in the LiP from T. cervina, playing the same function of the above catalytic tryptophan, is also indicated (blue square). Species abbreviations: AB, A. bisporus; B, Bjerkandera sp; BA, B. adusta; CC, C. cinerea; CD, C. disseminatus; CR, C. rivulosa; CS, C. subvermispora; DS, D. squalens; GA, G. applanatum; GAU, G. australe; GF, G. formosanum; IZU, unidentified basidiomycete IZU-154; LE, L. edodes; PC, P. chrysosporium; PE, P. eryngii; PO, P. ostreatus; PP, P. pulmonarius; PR, P. radiata; PS, P. sordida; PSA, P. sapidus; S, Spongipellis sp; TC, T. cervina; and TV, T. versicolor. Protein sequence entries: AB-MnP, CAG27835; B-VP, AAO47909; BA-LiP, 1906181A; CD-PerO, AAZ14938; CC-CIP, CAA50060; CR-MnPA, ABB83812; CR-MnPB, ABB83813; CS-MnP1, AAB03480; CS-MnP2, AAD43581; CS-MnP3, AAD45725; CS-MnP4, AAO61784; DS-MnP1, AAF31329; DS-MnP2, AAF31330; GA-MnP1, BAA88392; GAU-MnP, ABB77244; GF-MnP, ABB77243; IZU-MnP1, no entry available but sequence taken from the literature (Matsubara et al., 1996); LE-MnP1, BAE79199; PC-LiPA (isoenzyme H8), AAA53109; PC-LiPB (isoenzyme H8), AAA33741; PCLiPC (isoenzyme H10), AAA33739; PC-LiPD (isoenzyme H2), CAA33621; PC-LiPE (isoenzyme H8), AAA33738; PC-LiPF, AAA33736; PC-LiPH (isoenzyme H8), AAA56852; PC-LiPJ, AAD46494; PC-MnP1, AAA33744; PCMnP2, AAA33745; PC-MnP3, AAB39652; PC-NOPA, AAU82081; PE-VPL1, AAD01401; PE-VPL2, AAD01404; PE-VPL3, CAD56164 (and DQ056374); PE-VPS1, AAD54310; PO-MnP1, AAA84396; PO-MnP2, CAB51617; PO-MnP3, BAA33449; PP-MnP5, AAX40734; PR-LiP, P20010; PR-LiP1, AAW59419; PR-LiP4, AAW66483; PR-MnP2, CAC85963; PR-MnP3, CAC84573; PS-MnP1, BAC06185; PS-MnP2, BAC06186; PS-MnP3, BAC06187; PSA-VP, CAJ01576; S-MnP1, BAE79812; TC-LiP, AB191466; TV-LiP12, AAA34049; TV-LiP7, CAA83147; TV-LiPGII, CAA53333; TV-MnP2, CAA83148; TV-MRP, AAB63460; TV-PGV, CAA54398; and TV-PGVII, CAA91043. Versatile peroxidase VP oxidizes Mn2+, as MnP (EC 1.11.1.13) does, and also high redox-potential aromatic compounds, as LiP (EC 1.11.1.14) does. Due to their Mn-oxidizing activity, the Pleurotus VP isoenzymes were first described as MnP isoenzymes (Martínez et al., 1996; Giardina et al., 2000), but they were later recognized as representing a new peroxidase type (EC 1.11.1.16). VP is also able to efficiently oxidize phenolic compounds and dyes that are the substrates of generic peroxidases (EC 1.11.1.7), such as the C. cinerea peroxidase (CIP) (Baunsgaard et al., 1993) and related peroxidases, or the well-known horseradish peroxidase (HRP) (Veitch, 2004). By contrast, LiP is not able to oxidize phenolic compounds efficiently because of inactivation in the absence of veratryl alcohol (VA) or related substrates, and MnP only oxidize phenols in the presence of Mn2+, although a P. radiata short MnP seems to be an exception (Hildén et al., 2005). Moreover, VP directly oxidizes high redox-potential compounds, for example, the dye Reactive Black 5 (RB5), that LiP can oxidize only in the presence of redox mediators such as VA (Heinfling et al., 1998b). The Mn3+ formed by VP (and MnP) acts as a diffusible oxidizer on phenolic lignin and free phenols, as well as a starter of lipid peroxidation reactions involved in the biodegradation of recalcitrant compounds (Kapich et al., 2005). The VP family is included in Class II (fungal peroxidases) of the ‘plant, fungal, and bacterial peroxidase’ superfamily (Welinder, 1992) together with the MnP, LiP, and CIP families. The interest on VP has increased during the last years, both as a model enzyme and as a source of industrial/environmental biocatalysts. The molecular architecture of VP and other basidiomycete peroxidases includes a haem cofactor located at an internal cavity connected to the solvent by two access channels (Gold et al., 2000; Martínez, 2002). The main channel is conserved in all haem peroxidases and is used by hydrogen peroxide to reach and oxidize the haem cofactor forming the two-electron activated Compound I, which is stepwise reduced to Compound II and resting enzyme during two one-electron substrate oxidations. The second channel extends directly to the haem propionates being the site where MnP and VP oxidize Mn2+ to Mn3+, as described below. Bifunctional prokaryotic (Class I) ascorbate peroxidase also oxidizes ascorbate near the haem propionates (Sharp et al., 2003). Oxidation of phenolic compounds has been studied in peroxidases from Class III (plant peroxidases) and also from Class II, and the classical hypothesis that the reaction takes place in the vicinity of the haem has been demonstrated for plant HRP and fungal CIP (Smith and Veitch, 1998). However, no crystal structure of a ligninolytic peroxidase complexed with an aromatic substrate or analogue has been reported to date. Moreover, it is generally accepted that the main haem channel, in spite of certain degree of plasticity (Gerini et al., 2003), is too narrow for direct contact between the haem cofactor and most aromatic substrates including lignin. Therefore, long-range electron transfer (LRET) from a protein radical at the surface of the enzyme, which would act as the substrate oxidizer, to the haem cofactor has been suggested to explain oxidation of aromatic substrates, redox mediators, and even the lignin polymer. As discussed below, dual oxidation of some aromatic substrates and dyes at the VP surface (by LRET) and at the haem channel is considered in the light of some recent results. Mn-oxidation site in VP Enzymatic oxidation of Mn2+ to Mn3+ is a unique characteristic of basidiomycete MnP and VP, although it has also been reported in prokaryotic catalase-peroxidase (Magliozzo and Marcinkeviciene, 1997). The Mn2+-oxidation site in P. eryngii VP is similar to that of P. chrysosporium MnP (Gold et al., 2000; Sundaramoorthy et al., 2005), but it has certain differences as revealed by crystallographic, site-directed mutagenesis, and kinetic studies (Ruiz-Dueñas et al., 2007b). The Mn2+-binding site in VP is formed by the side-chains of Glu36, Glu40, and Asp175 located in front of the internal (i.e. more distant from the main haem access-channel) propionate of haem (Fig. 2, top), and connected to the solvent by a narrow access-channel that presents a variable geometry during catalysis. The carboxylates of the above amino-acid residues and the haem propionate are responsible of Mn2+ binding for subsequent electron transfer to the activated haem of VP Compounds I and II. Fig. 2. View largeDownload slide Axial views of the haem region showing the exposed catalytic tryptophan in VP (Trp164) and LiP (Trp171) responsible for oxidation of high redox-potential substrates by LRET to haem (red arrow), and the residues forming the Mn2+-binding site near the internal propionate of haem in VP (Glu36, Glu40, and Asp175) and MnP (Glu35, Glu39, and Asp179). Mn2+ is shown as a van der Waals sphere, and several water molecules are included (red spheres). From PDB entries, 2BOQ, 1YYD, and 1LLP for VP, MnP, and LiP, respectively. Fig. 2. View largeDownload slide Axial views of the haem region showing the exposed catalytic tryptophan in VP (Trp164) and LiP (Trp171) responsible for oxidation of high redox-potential substrates by LRET to haem (red arrow), and the residues forming the Mn2+-binding site near the internal propionate of haem in VP (Glu36, Glu40, and Asp175) and MnP (Glu35, Glu39, and Asp179). Mn2+ is shown as a van der Waals sphere, and several water molecules are included (red spheres). From PDB entries, 2BOQ, 1YYD, and 1LLP for VP, MnP, and LiP, respectively. The crystal structures (solved up to 1.3 Å resolution) of wild and Escherichia coli (Migula) Castellani & Chalmers expressed VP showed a variable orientation of the Glu36 and Glu40 side-chains, whereas that of Asp175 is maintained (Ruiz-Dueñas et al., 2007b). The position in the in vitro activated recombinant VP (Pérez-Boada et al., 2002) before its exposure to Mn2+ (and in the Mn2+ exposed D175A variant, which suffer from a strong decrease in the ability to bind and oxidize the cation) corresponds to an ‘open-gate’ conformation enabling Mn2+ access to the oxidation site. The conformation in which the two glutamate side-chains point toward the Mn2+ ion correspond to the ‘closed-gate’ conformation, and was found in the naturally-folded enzyme from Mn2+-containing P. eryngii cultures. In this conformation Glu36, Glu40, Asp175, and propionate carboxylates are at co-ordination distance from Mn2+. In contrast to that reported for MnP (Sundaramoorthy et al., 1994, 1997), the recombinant VP crystals grown in the presence of Mn2+ were unstable, and the addition of Zn2+ to grow crystals after Mn2+ exposure resulted in an intermediate conformation of the metal binding site, with the Glu40 side-chain at a position different from that found in wild VP crystals. The ‘closed-gate’ conformation described above coincides with that observed in P. chrysosporium MnP crystallized in the presence of Mn2+, where three homologous residues (Glu35, Glu39, and Asp179) participate in metal co-ordination (Sundaramoorthy et al., 1994) (Fig. 2, centre). Opening of the glutamate side-chains was not observed in MnP crystals grown in the absence of Mn2+ (Sundaramoorthy et al., 1997). However, displacement of Glu35 and Glu39 side-chains was found in crystals of a mutated MnP variant that does not bind Mn2+ (Sundaramoorthy et al., 1997) and in MnP crystals where the inhibitor Sm3+ was removed from the metal binding site (Sundaramoorthy et al., 2005). Additional amino acid residues, which are absent in VP, contribute to orientation of the Mn2+ ligands in MnP affecting their mobility. This is the case of Arg177 forming a salt bridge with Glu35 necessary for the optimal Mn2+ co-ordination and ligation geometry in MnP (Gelpke et al., 2000) Site-directed mutagenesis was performed to evaluate the involvement of the three above acidic residues in VP catalysis (Ruiz-Dueñas et al., 2007b). The E36A, E40A, and D175A mutations caused 4–20-fold decrease in Mn2+ oxidation activity (kcat) under steady-state conditions (characterized by VP saturating concentrations of both oxidizing and reducing substrates). Moreover, the selectivity constant (kcat/Km, also known as catalytic efficiency) was 250–1250-fold decreased, indicating that the above residues are responsible for selective binding of Mn2+ near the haem. The decreases in VP selectivity constant were much lower than found in similar MnP variants (E40A and D175A) (Kishi et al., 1996; Whitwam et al., 1997; Youngs et al., 2001). This is due to differences in kcat, which decreased from similar values in both native enzymes (around 300 s−1) to much lower values in the MnP variants (1–3 s−1) than in the VP variants (15–85 s−1). In fact, removal of the three carboxylates in a triple variant (E36A/E40A/D175A) was required to lower the VP activity to levels (<1% kcat) similar to those found in MnP single variants. Transient-state kinetic constants showed that reduction of both VP Compounds I and II by Mn2+ (estimated from changes of their characteristic spectra using stopped-flow rapid spectrophotometry) was modified by the E36A, E40A, and D175A mutations. The apparent second-order rate constants revealed that reduction of Compound II was much more affected (103–104-fold lower k3app) than Compound I reduction (80–325-fold lower k2app), the former being a rate-limiting step in VP catalysis. Site-directed mutagenesis of P. chrysosporium MnP also showed that reduction of both Compound II (Kusters-van Someren et al., 1995; Kishi et al., 1996) and Compound I (Whitwam et al., 1997; Youngs et al., 2001) by Mn2+ was lowered by mutations at the binding site. However, the decreases of apparent second-order rate constants were lower in the VP than in the homologous MnP variants. Moreover, the first-order constants for Compound II reduction (Compound I reduction is too fast to be measured) showed a strong increase of the dissociation constant (300–1000-fold), but only slightly affected k3 (2–30-fold decrease) in the case of VP mutants, whereas a strong decrease in k3 (100–1200-fold) was also observed in the corresponding MnP variants. The selectivity constant for Mn2+ also decreased (50–75-fold) when the acidic side-chains were shortened in the E36D and E40D variants that, nevertheless, retained 30–50% of their steady-state activity (kcat). This strongly contrasts with the behaviour of P. chrysosporium MnP, where similar mutations (E35D and E39D) caused 50–100 fold decrease of kcat (Kishi et al., 1996; Youngs et al., 2001). The same tendency was observed in the first-order rate constant for Compound II reduction (k3) that showed 40–120-fold lower values in the MnP variants but only a 1–4-fold decrease in the VP variants. The kinetic constants of VP mutated variants at Glu36 and Glu40 positions showed that the Mn2+-oxidation site of VP, although having the same Mn-co-ordinating residues as found in MnP, operates in a slightly different way, in the sense that Mn2+ is still efficiently oxidized when one of the three amino acid carboxylates is removed (Glu/Asp→Ala mutations) or when the length of the acidic side-chains is reduced (Glu→Asp mutations) resulting in much higher kcat and k3 values than found for homologous MnP variants. A putative MnP has been reported in Ganoderma applanatum (Pers.) Pat., Ganoderma australe (Fr.) Pat., and Ganoderma formosanum T.T. Chang & T. Chen (Maeda et al., 2001) with only two acidic residues at the putative Mn2+-oxidation site. However, the catalytic properties of these peroxidases should be re-examined, since sequence alignment showed a tryptophan residue homologous to the VP Trp164 (or LiP Trp171) discussed below (Fig. 1). Mn-oxidation sites (red circles in Fig. 1) have been identified in the sequences (and molecular models when available) of 23 MnP and six VP, as well as in five putative VP that were initially described as MnP, including MnP1 from P. ostreatus and MnP5 from Pleurotus pulmonarius (Fr.) Quel., or LiP (and one of them as an unclassified Mn-repressed peroxidase). The kinetic constants of these putative VP on representative substrates should be re-examined. VP oxidation of high redox-potential substrates: LRET mechanism LRET occurs in different redox proteins, like prokaryotic cytochrome-c peroxidase that oxidizes cytochrome c at the protein surface, and transfers electrons to a stable tryptophan radical near the haem cofactor via three amino acid residues (Pelletier and Kraut, 1992). LRET has been suggested to be involved in lignin biodegradation by ligninolytic haem peroxidases for electron transfer both: (i) in the peroxidase molecule, to overcome the steric hindrances preventing direct interaction between the haem group and the lignin polymer; and (ii) in the lignin macromolecule itself resulting in breakdown of the most labile inter-unit linkages (Schoemaker et al., 1994a, b). Crystal structures and homology models of two P. eryngii VP isoenzymes revealed three possible LRET pathways for the oxidation of high redox-potential aromatic compounds (Camarero et al., 1999; Ruiz-Dueñas et al., 1999; Pérez-Boada et al., 2005). They would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 (homologous to VPL Trp164) of isoenzyme VPS1. These residues are exposed, and less than 11 Å apart from the haem. The corresponding pathways would be similar to those proposed for P. chrysosporium LiP that would start at His82, Trp171, or His239 (Du et al., 1992; Schoemaker et al., 1994a; English et al., 1995; Johjima et al., 1999). With the purpose of investigating the functionality of these three putative pathways, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways present in this isoenzyme; and (ii) incorporated the absent pathway (Pérez-Boada et al., 2005). The steady-state kinetic constants of the variants obtained definitively showed that Trp164 (see Fig. 2, top) is required for the oxidation of the two high redox-potential model substrates investigated, VA and RB5, whereas the two other putative pathways (starting at His232 and His82) did not seem to be involved in catalysis. Transient-state kinetics showed that the W164S mutation completely blocked the reduction of Compound II by both substrates, whereas the apparent second-order rate constant for Compound I reduction was only partially decreased (15–50-fold lower k2app values) (Ruiz-Dueñas et al., 2008a). The above results were in agreement with those obtained by mutagenesis of homologous Trp171 (Fig. 2, bottom) and His82 in P. chrysosporium LiP (mutagenesis of His239 has not been reported) (Doyle et al., 1998; Mester et al., 2001a; Gelpke et al., 2002) and contrasted with some unconfirmed results suggesting that VA could be oxidized at the haem channel (Ambert-Balay et al., 1998). Involvement of a tryptophan residue in VP catalysis had been suggested by N-bromosuccinimide bromination of Pleurotus and Bjerkandera VP (Ayala-Aceves et al., 2001; Ruiz-Dueñas et al., 2001; Kamitsuji et al., 2005; Pogni et al., 2005). In addition to the site-directed mutagenesis of P. eryngii VP described above, mutagenesis of the catalytic tryptophan of P. ostreatus VP has recently been reported using a homologous expression system (Tsukihara et al., 2008). It has also been shown that a tetrameric lignin model compound is degraded by LiP but it is not oxidized by its W171S variant (Mester et al., 2001a). This suggests that polymeric lignin would be oxidized at the same exposed tryptophan involved in VA oxidation. However, this conclusion requires additional investigation, since it has also been reported that a lignin dehydrogenation polymer would bind LiP at His239 (Johjima et al., 1999). Concerning the aromatic substrate oxidation mechanism, Pérez-Boada et al. (2005) provided the first direct evidence on the formation of a protein (Trp164) radical in a ligninolytic peroxidase using low-temperature electron paramagnetic resonance (EPR) of H2O2-activated P. eryngii VP. However, indirect evidence on the formation of a Trp171 radical had already been reported in LiP (Blodig et al., 1999). This is in agreement with EPR results obtained with B. adusta VP (Pogni et al., 2005), but contrasted with unconfirmed LiP studies that assigned the Compound I EPR signal to a porphyrin π cation radical (Khindaria and Aust, 1996). Additional spectroscopic and density functional theory studies confirmed the location of the tryptophan radical in VP, and showed that it exists in the neutral form (Pogni et al., 2006, 2007). Substitution of Trp164 either by serine or histidine resulted in similar incapability of both variants to oxidize high redox-potential compounds, indicating that the imidazole side-chain is not playing a similar role as the indole side-chain. Interestingly, a LiP-type peroxidase has been reported in T. cervina that has an exposed tyrosine residue involved in catalysis, although located at a different position than the LiP and VP catalytic tryptophan (Miki et al., 2006). However, the W164Y variant of VP was not catalytically-active on VA and RB5 under steady-state conditions due to the blocked reduction of Compound II, although the corresponding tyrosinyl radical was clearly detected by EPR (Ruiz-Dueñas et al., 2008c). A significant difference between VP and P. chrysosporium LiP concerns autocatalytic modification of LiP Trp171. It has been reported that Trp171 in P. chrysosporium LiP is naturally β-hydroxylated, most probably due to peroxide excess (with respect to reducing substrate) in fungal cultures (Blodig et al., 1998, 2001). However, this is not the case in VP as shown by X-ray diffraction of crystals from both wild and peroxide-treated recombinant VP (Pérez-Boada et al., 2005). The origin and physiological significance of this difference is to be determined. Exposed tryptophan residues homologous to VP Trp164 (blue circles in Fig. 1) have been identified in the sequences (and molecular models when available) of 14 LiP and six VP, as well as in three putative LiP and five putative VP, the latter already mentioned above. The kinetic constants of these putative LiP (and VP) on veratryl alcohol and other substrates should be re-examined. VP oxidation of high redox-potential substrates: catalytic Trp164 environment Some characteristics of the surface environment of the catalytic tryptophan (VP Trp164 and LiP Trp171) could also be at the origin of differences in the oxidation of high redox-potential aromatic substrates, such as the need for redox mediators in LiP oxidation of some compounds that are directly oxidized by VP (e.g. RB5), and the lower VP efficiency oxidizing VA. With the purpose of investigating this hypothesis, different residues near the Trp164 of VP were modified by site-directed mutagenesis (Ruiz-Dueñas et al., 2008a). Some of the mutations strongly modified the kinetics of VA and RB5 oxidation by VP, demonstrating that other residues at the catalytic tryptophan environment contribute to substrate oxidation by ligninolytic peroxidases. The most outstanding characteristic of the LiP surface around the catalytic Trp171 is the protruding side-chain of Phe267 in an environment with a partial negative charge, whereas VP has several basic residues in the Trp164 environment, and Ala260 at the position of LiP Phe267 (Ruiz-Dueñas et al., 2008a). It was first considered by the authors that the lack of direct activity of LiP on RB5 could be due to the presence of Phe267 interrupting the furrow where this large substrate would be accommodated in VP. However, this did not seem to be the case since introduction of a phenylalanine residue (A260F variant) did not modify RB5 oxidation by VP. By contrast, VP activity on RB5 was eliminated by the R257D mutation. Therefore, the current hypothesis is that the lack of LiP activity on RB5 would not be due to the steric hindrances mentioned above, and also suggested by other authors (Tsukihara et al., 2008), but to the existence of a partially-negative environment (including Asp264 homologous to VP Arg257, and Asp165 homologous to VP Ser158) compared with the positively-charged environment of VP Trp164 (including Lys264 in addition to Arg257) that would favour binding of anionic substrates. On the other hand, it was demonstrated that three of the five exposed acidic residues around Trp171 in P. chrysosporium LiP affected the kinetics of VA oxidation (Smith and Doyle, 2006) and suggested that a partially acidic environment would stabilize the VA cation radical (VA•+) formed to act as an enzyme-bond mediator (Khindaria et al., 1997). Two of the acidic residues in LiP (Glu168 and Glu250) are conserved in VP, and the three others were introduced as single and/or multiple VP mutations. In contrast with the initial expectations that predicted a more efficient oxidation of VA, the N256D/R257D/A260F VP variant completely lost its activity on VA. Moreover, this and other variants bearing the R257D, N256D or S158E mutations also lost their activity on RB5. Interestingly, the RB5-oxidizing activity of the R257D variant was restored by adding VA to the reaction mixture. This suggested stabilization of VA•+ by a more acidic Trp164 environment in the mutated VP, similar to that found in LiP. Such behaviour is similar to the VA-mediated activity of LiP on RB5 and other substrates, and contrasted with RB5 direct oxidation by native VP (Heinfling et al., 1998b). Finally, transient-state kinetic studies revealed that combination of the A260F and R257A mutations in a VP double variant strongly increased the apparent second-order rate constants for the reduction of Compounds I and II by VA (over 20-fold and 50-fold improvement, respectively). Moreover, the first-order rate and dissociation constants for Compound II reduction showed improvement of both the k3 and KD3 constants. It is noteworthy that the k3app for reduction of Compound II of the R257A/A260F variant by VA (68 mM−1 s−1) was only slightly lower than reported for P. chrysosporium LiP (185 mM−1 s−1); and its k3 (46 s−1) was even higher than that of LiP (34 s−1) (Gelpke et al., 2002). The involvement of Phe267 in VA binding had been reported in LiP (Gelpke et al., 2002). The VP A260F and R257L single variants also showed some decreases of VA KD3, but the k3 values were not comparable to that of the VP double variant, indicating that the simultaneous removal of Arg257 and incorporation of a phenylalanine residue (homologous to LiP Phe267) are required to obtain a VP variant with VA transient-state kinetic constants comparable to those of LiP. It has been suggested that the discrepancy between the strongly improved transient-state constants and the barely modified steady-state constants of the R257/A260F variant of VP could be due to slow dissociation of the enzyme–product complex (Ruiz-Dueñas et al., 2008a). However, if VA•+ is the product of VA oxidation by VP and LiP, and veratraldehyde is formed by subsequent reaction with O2 (Schoemaker et al., 1994b), the latter reaction could also be the rate-limiting step in veratraldehyde formation by this variant. Oxidation of low redox-potential substrates at the VP main haem channel Sigmoidal curves are obtained for oxidation of different concentrations of low redox-potential 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS), p-hydroquinone, and 2,6-dimethoxyphenol by VP (Heinfling et al., 1998b; Ruiz-Dueñas et al., 2001) enabling the calculation of two sets of Km and kcat constants. This double kinetics suggested two independent oxidation sites, characterized by high and low specificity constants. This was confirmed by site-directed mutagenesis of VP Trp164 resulting in the removal of the high-specificity site for ABTS oxidation, whereas the low-specificity site remained (Ruiz-Dueñas et al., 2008a). Recently, a similar effect of the W164S mutation has been found on the VP oxidation of phenols. Simultaneously, the location of the low specificity oxidation site has been identified by site-directed mutagenesis at the main haem access channel. This resulted in several-fold increased specificity constants for phenols after removing the side-chains of some amino acids increasing the channel size (Ruiz-Dueñas et al., 2008b). To confirm substrate oxidation at the haem channel in these improved variants, the mutations opening the haem channel were combined with the W164S mutation. Variants with specificity constants similar to those of the high specificity site in native VP were obtained in spite of the removal of Trp164. These studies provided evidence on the oxidation of different substrates at the main haem channel of native VP, albeit with a lower specificity than observed at the catalytic Trp164 (that, in addition, is able to oxidize high redox-potential substrates that are not oxidized at the haem channel of native VP). This third substrate-oxidation site in VP is similar to the classical oxidation site reported in different peroxidases, including CIP (Smith et al., 1998; Tsukamoto et al., 1999). Interestingly, its operation is improved when the haem channel in VP, which is smaller than in CIP and plant peroxidases, is opened by site-directed mutagenesis. Studies currently being conducted include transient-state kinetics and crystallographic studies of these haem channel variants. It is possible to conclude that the versatile catalytic properties of P. eryngii VP are due to a hybrid molecular architecture that includes in the same protein different oxidation sites (for Mn2+, high redox-potential aromatics, and low redox-potential phenols and dyes) related to those found in the other basidiomycete peroxidase families (MnP, LiP, and CIP, respectively). Biotechnological interest of VP and other basidiomycete peroxidases Xylanases were introduced in the bleaching plant of paper-pulp mills, as a substitute for chlorine-containing reagents (Viikari et al., 1994), at a time when barely any information on ligninolytic peroxidases was available. However, enzymes acting on lignin (ligninolytic peroxidases and laccases) are the biocatalysts of choice for environmentally sound bleaching (Bajpai, 2004; Sigoillot et al., 2005). Laccases have been known for decades and are already produced in high yields, however, ligninolytic peroxidases are not commercially available despite the considerable efforts devoted to improve their heterologous expression (Conesa et al., 2000, 2002; Gu et al., 2003; Lú-Chau et al., 2004; Eibes et al., 2008). Laccases have low redox-potential and can degrade lignin only in the presence of mediators, whose costs and potential environmental risks are the main obstacles for implementing the laccase-mediator systems at the mill scale. By contrast, some ligninolytic peroxidases do not require mediators to degrade high redox-potential compounds. Among the different basidiomycete peroxidases, VP presents particular interest due to its catalytic versatility including the degradation of compounds that other peroxidases are not able to oxidize directly. VP versatility permits its application in Mn3+-mediated or Mn-independent reactions on both low and high redox-potential aromatic substrates and dyes, among others. It has been shown recently that VP can be used to reoxidize Mn-containing polyoxometalates, which are efficient oxidizers in paper pulp delignification (Marques et al., 2008). Another aspect of VP catalysis, which has been reported recently and merits investigation in more detail for some applications, is its high activity in the presence of organic solvents (Rodakiewicz-Nowak et al., 2006). The ability to degrade directly a variety of recalcitrant compounds represents a VP characteristic of the highest biotechnological interest. The use of aromatic mediators acting via their cation radicals (Schoemaker et al., 1994b) is required for LiP oxidation of simple phenols (Koduri and Tien, 1995), monomethoxylated aromatics (Harvey et al., 1986), polymeric dyes including Poly R-478 (Harvey et al., 1995; Paszczynski and Crawford, 1991), PAH (Barr and Aust, 1994), high redox-potential dyes (Heinfling et al., 1998b), ferrocytochrome c (Sheng and Gold., 1998), and, most important, polymeric lignin (Hammel et al., 1993). By contrast, VP does not requires mediators to oxidize many of the above compounds, including phenolic and non-phenolic aromatic pollutants (Rodríguez et al., 2004), pesticides (Dávila-Vázquez et al., 2005), high redox-potential and polymeric dyes (Heinfling et al., 1998a; Kamitsuji et al., 2005; Tinoco et al., 2007), PAH (Wang et al., 2003), and lignin (Camarero et al., 2001; Moreira et al., 2007). This VP ability represents an important advantage with respect to LiP, since the cost of VA as a mediator can be saved. Moreover, for more efficient oxidation of some recalcitrant compounds VP can also be used in combination with redox mediators including natural phenolic compounds (Tinoco et al., 2007). The abundant information on peroxidase structure–function relationships currently available has already been used to modulate the catalytic and operational properties of these enzymes (including peroxide inactivation resistance) as industrial biocatalysts using site-directed mutagenesis (Yeung et al., 1997; Wilcox et al., 1998; Timofeevski et al., 1999; Reading and Aust, 2000; Celik et al., 2001; Mester and Tien, 2001b; Miyazaki and Takahashi, 2001; Valderrama et al., 2002; Feng et al., 2003; Ryan et al., 2006; Pfister et al., 2007; Ruiz-Dueñas et al., 2008b). In those cases where the structural basis of the property to be improved is unknown, or too difficult to be predicted, directed evolution and/or combinatorial mutagenesis are the approach of choice (Cherry et al., 1999; Miyazaki-Imamura et al., 2003; Ryu et al., 2008). It is expected that genetic engineering will also contribute to overcoming the main drawback for the industrial utilization of VP and other ligninolytic peroxidases, i.e. their low yields in heterologous expression. An alternative approach is to engineer already commercial (i.e. high-yield expressed) peroxidases by introducing the ability to oxidize high-redox potential compounds by site-directed mutagenesis, based on the structure–function information already available. Using a combination of site-directed mutagenesis, saturation mutagenesis, directed evolution, and other molecular biology techniques, tailor-made biocatalysts will be available in the future enabling industrial exploitation of the unique catalytic abilities of the high redox-potential peroxidases (VP, LiP, and MnP) produced by the lignin-degrading basidiomycetes. Abbreviations Abbreviations ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) CIP Coprinopsis cinerea peroxidase EPR electron paramagnetic resonance MnP manganese peroxidase LiP lignin peroxidase LRET long-range electron transfer PAH polycyclic aromatic hydrocarbons RB5 Reactive Black 5 VA veratryl alcohol VA•+ VA cation radical VP versatile peroxidase This work was supported by the Spanish project BIO2005-03569 and BIO2008-01533, and the BIORENEW EU-project (NMP2-CT-2006-026456). The authors thank Klaus Piontek (Albert-Ludwigs University of Freiburg, Germany), María J Mate (CIB, CSIC, Madrid), and Antonio Romero (CIB, CSIC, Madrid) for crystallographic data; Rebecca Pogni and Riccardo Basosi (University of Sienna, Italy) for EPR analyses; and Andrew T Smith (University of Sussex, Brighton, UK) for helpful comments. FJR-D, EG, and YM thank the EU for a project contract, and MM thanks CSIC for an I3P fellowship. References Ambert-Balay K,  Fuchs SM,  Tien M.  Identification of the veratryl alcohol binding site in lignin peroxidase by site-directed mutagenesis,  Biochemistry and Biophysics Research Communications ,  1998, vol.  251 (pg.  283- 286) Google Scholar CrossRef Search ADS   Ayala-Aceves M,  Baratto MC,  Basosi R,  Vázquez-Duhalt R,  Pogni R.  Spectroscopic characterization of a manganese–lignin peroxidase hybrid isozyme produced by Bjerkandera adusta in the absence of manganese: evidence of a protein-centred radical by hydrogen peroxide,  Journal of Molecular Catalysis B: Enzymatic ,  2001, vol.  16 (pg.  159- 167) Google Scholar CrossRef Search ADS   Bajpai P.  Biological bleaching of chemical pulps,  Critical Reviews in Biotechnology ,  2004, vol.  24 (pg.  1- 58) Google Scholar CrossRef Search ADS PubMed  Barr DP,  Aust SD.  Mechanisms white rot fungi use to degrade pollutants,  Environmental Science and Technology ,  1994, vol.  28 (pg.  A78- A87) Google Scholar CrossRef Search ADS   Baunsgaard L,  Dalboge H,  Houen G,  Rasmussen EM,  Welinder KG.  Amino acid sequence of Coprinus macrorhizus peroxidase and cDNA sequence encoding Coprinus cinereus peroxidase: a new family of fungal peroxidases,  European Journal of Biochemistry ,  1993, vol.  213 (pg.  605- 611) Google Scholar CrossRef Search ADS PubMed  Blodig W,  Doyle WA,  Smith AT,  Winterhalter K,  Choinowski TH,  Piontek K.  Autocatalytic formation of hydroxy group at Cβ of Trp171 in lignin peroxidase,  Biochemistry ,  1998, vol.  37 (pg.  8832- 8838) Google Scholar CrossRef Search ADS PubMed  Blodig W,  Smith AT,  Doyle WA,  Piontek K.  Crystal structures of pristine and oxidatively processed lignin peroxidase expressed in Escherichia coli and of the W171F variant that eliminates the redox active tryptophan 171. Implications for the reaction mechanism,  Journal of Molecular Biology ,  2001, vol.  305 (pg.  851- 861) Google Scholar CrossRef Search ADS PubMed  Blodig W,  Smith AT,  Winterhalter K,  Piontek K.  Evidence from spin-trapping for a transient radical on tryptophan residue 171 of lignin peroxidase,  Archives of Biochemistry and Biophysics ,  1999, vol.  370 (pg.  86- 92) Google Scholar CrossRef Search ADS PubMed  Bourbonnais R,  Paice MG.  Oxidation of non-phenolic substrates. An expanded role for laccase in lignin biodegradation,  FEBS Letters ,  1990, vol.  267 (pg.  99- 102) Google Scholar CrossRef Search ADS PubMed  Camarero S,  Bocchini P,  Galletti GC,  Martínez MJ,  Martínez AT.  Compositional changes of wheat lignin by a fungal peroxidase analyzed by Pyrolysis-GC/MS,  Journal of Analytical and Applied Pyrolysis ,  2001, vol.  58/59 (pg.  413- 423) Google Scholar CrossRef Search ADS   Camarero S,  Ibarra D,  Martínez MJ,  Martínez AT.  Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes,  Applied and Environmental Microbiology ,  2005, vol.  71 (pg.  1775- 1784) Google Scholar CrossRef Search ADS PubMed  Camarero S,  Sarkar S,  Ruiz-Dueñas FJ,  Martínez MJ,  Martínez AT.  Description of a versatile peroxidase involved in natural degradation of lignin that has both Mn-peroxidase and lignin-peroxidase substrate binding sites,  Journal of Biological Chemistry ,  1999, vol.  274 (pg.  10324- 10330) Google Scholar CrossRef Search ADS PubMed  Celik A,  Cullis PM,  Sutcliffe MJ,  Sangar R,  Raven EL.  Engineering the active site of ascorbate peroxidase,  European Journal of Biochemistry ,  2001, vol.  268 (pg.  78- 85) Google Scholar CrossRef Search ADS PubMed  Cherry JR,  Lamsa MH,  Schneider P,  Vind J,  Svendsen A,  Jones A,  Pedersen AH.  Directed evolution of a fungal peroxidase,  Nature Biotechnology ,  1999, vol.  17 (pg.  379- 384) Google Scholar CrossRef Search ADS PubMed  Conesa A,  Jeenes D,  Archer DB,  vandenHondel CAMJ,  Punt PJ.  Calnexin overexpression increases manganese peroxidase production in Aspergillus niger,  Applied and Environmental Microbiology ,  2002, vol.  68 (pg.  846- 851) Google Scholar CrossRef Search ADS PubMed  Conesa A,  van den Hondel CAMJJ,  Punt PJ.  Studies on the production of fungal peroxidases in Aspergillus niger,  Applied and Environmental Microbiology ,  2000, vol.  66 (pg.  3016- 3023) Google Scholar CrossRef Search ADS PubMed  Cullen D,  Kersten PJ.  Brambl R,  Marzluf GA.  Enzymology and molecular biology of lignin degradation,  Mycota III. Biochemistry and molecular biology ,  2004 Berlin Springer(pg.  249- 273) Dávila-Vázquez G,  Tinoco R,  Pickard MA,  Vázquez-Duhalt R.  Transformation of halogenated pesticides by versatile peroxidase from Bjerkandera adusta,  Enzyme and Microbial Technology ,  2005, vol.  36 (pg.  223- 231) Google Scholar CrossRef Search ADS   Doyle WA,  Blodig W,  Veitch NC,  Piontek K,  Smith AT.  Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis,  Biochemistry ,  1998, vol.  37 (pg.  15097- 15105) Google Scholar CrossRef Search ADS PubMed  Du P,  Collins JR,  Loew GH.  Homology modeling of a haem protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase,  Protein Engineering ,  1992, vol.  5 (pg.  679- 691) Google Scholar CrossRef Search ADS PubMed  Eibes GM,  Lú-Chau TA,  Ruiz-Dueñas FJ,  Feijoo G,  Martínez MJ,  Martínez AT,  Lema JM.  Effect of culture temperature on the heterologous expression of Pleurotus eryngii versatile peroxidase in Aspergillus hosts,  Bioprocesses and Biosystems Engineering ,  2008  DOI 10.1007/s00449-008-0231-7 English AM,  Tsaprailis G.  Catalytic and structure-function relationships in haem proteins,  Advances in Inorganic Chemistry ,  1995, vol.  43 (pg.  79- 125) Feng ML,  Tachikawa H,  Wang XT,  Pfister TD,  Gengenbach AJ,  Lu Y.  Resonance Raman spectroscopy of cytochrome c peroxidase variants that mimic manganese peroxidase,  Journal of Biological Inorganic Chemistry ,  2003, vol.  8 (pg.  699- 706) Google Scholar CrossRef Search ADS PubMed  Gelpke MDS,  Lee J,  Gold MH.  Lignin peroxidase oxidation of veratryl alcohol: effects of the mutants H82A, Q222A, W171A, and F267L,  Biochemistry ,  2002, vol.  41 (pg.  3498- 3506) Google Scholar CrossRef Search ADS PubMed  Gelpke MDS,  Youngs HL,  Gold MH.  Role of arginine 177 in the MnII binding site of manganese peroxidase. Studies with R177D, R177E, R177N, and R177Q mutants,  European Journal of Biochemistry ,  2000, vol.  267 (pg.  7038- 7045) Google Scholar CrossRef Search ADS PubMed  Gerini MF,  Roccatano D,  Baciocchi E,  Di Nola A.  Molecular dynamics simulations of lignin peroxidase in solution,  Biophysical Journal ,  2003, vol.  84 (pg.  3883- 3893) Google Scholar CrossRef Search ADS PubMed  Giardina P,  Palmieri G,  Fontanella B,  Rivieccio V,  Sannia G.  Manganese peroxidase isoenzymes produced by Pleurotus ostreatus grown on wood sawdust,  Archives of Biochemistry and Biophysics ,  2000, vol.  376 (pg.  171- 179) Google Scholar CrossRef Search ADS PubMed  Gold MH,  Youngs HL,  Gelpke MD.  Manganese peroxidase,  Metal Ions in Biological Systems ,  2000, vol.  37 (pg.  559- 586) Google Scholar PubMed  Gu L,  Lajoie C,  Kelly C.  Expression of a Phanerochaete chrysosporium manganese peroxidase gene in the yeast Pichia pastoris,  Biotechnology Progress ,  2003, vol.  19 (pg.  1403- 1409) Google Scholar CrossRef Search ADS PubMed  Guillén F,  Evans CS.  Anisaldehyde and veratraldehyde acting as redox cycling agents for H2O2 production by Pleurotus eryngii,  Applied and Environmental Microbiology ,  1994, vol.  60 (pg.  2811- 2817) Google Scholar PubMed  Hammel KE,  Cullen D.  Role of fungal peroxidases in biological ligninolysis,  Current Opinion in Plant Biology ,  2008, vol.  11 (pg.  349- 355) Google Scholar CrossRef Search ADS PubMed  Hammel KE,  Jensen KA,  Mozuch MD,  Landucci LL,  Tien M,  Pease EA.  Ligninolysis by a purified lignin peroxidase,  Journal of Biological Chemistry ,  1993, vol.  268 (pg.  12274- 12281) Google Scholar PubMed  Harvey PJ,  Candeias LP,  King PJ,  Palmer JM,  Wever R.  Lignin peroxidase catalysis: Reaction with veratryl alcohol and a polymeric dye, Poly R,  Biochemistry Society Transactions ,  1995, vol.  23 pg.  S340  Google Scholar CrossRef Search ADS   Harvey PJ,  Schoemaker HE,  Palmer JM.  Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium,  FEBS Letters ,  1986, vol.  195 (pg.  242- 246) Google Scholar CrossRef Search ADS   Heinfling A,  Martínez MJ,  Martínez AT,  Bergbauer M,  Szewzyk U.  Transformation of industrial dyes by manganese peroxidase from Bjerkandera adusta and Pleurotus eryngii in a manganese-independent reaction,  Applied and Environmental Microbiology ,  1998, vol.  64 (pg.  2788- 2793) Google Scholar PubMed  Heinfling A,  Ruiz-Dueñas FJ,  Martínez MJ,  Bergbauer M,  Szewzyk U,  Martínez AT.  A study on reducing substrates of manganese-oxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta,  FEBS Letters ,  1998, vol.  428 (pg.  141- 146) Google Scholar CrossRef Search ADS PubMed  Higuchi T.  Microbial degradation of lignin: role of lignin peroxidase, manganese peroxidase, and laccase,  Proceedings of the Japanese Academy B ,  2004, vol.  80 (pg.  204- 214) Google Scholar CrossRef Search ADS   Hildén K,  Martínez AT,  Hatakka A,  Lundell T.  The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent,  Fungal Genetics and Biology ,  2005, vol.  42 (pg.  403- 419) Google Scholar CrossRef Search ADS PubMed  Hofrichter M,  Ullrich R.  Haem-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance,  Applied Microbiology and Biotechnology ,  2006, vol.  71 (pg.  276- 288) Google Scholar CrossRef Search ADS PubMed  Johjima T,  Itoh H,  Kabuto M,  Tokimura F,  Nakagawa T,  Wariishi H,  Tanaka H.  Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium,  Proceedings of the National Academy of Sciences, USA ,  1999, vol.  96 (pg.  1989- 1994) Google Scholar CrossRef Search ADS   Kamitsuji H,  Watanabe T,  Honda Y,  Kuwahara M.  Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isozyme from Pleurotus ostreatus without redox mediators,  Biochemical Journal ,  2005, vol.  386 (pg.  387- 393) Google Scholar CrossRef Search ADS PubMed  Kapich AN,  Steffen KT,  Hofrichter M,  Hatakka A.  Involvement of lipid peroxidation in the degradation of a non-phenolic lignin model compound by manganese peroxidase of the litter-decomposing fungus Stropharia coronilla,  Biochemical and Biophysical Research Communications ,  2005, vol.  330 (pg.  371- 377) Google Scholar CrossRef Search ADS PubMed  Kersten P,  Cullen D.  Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium,  Fungal Genetics and Biology ,  2007, vol.  44 (pg.  77- 87) Google Scholar CrossRef Search ADS PubMed  Kersten PJ,  Kirk TK.  Involvement of a new enzyme, glyoxal oxidase, in extracellular H2O2 production by Phanerochaete chrysosporium,  Journal of Bacteriology ,  1987, vol.  169 (pg.  2195- 2201) Google Scholar PubMed  Khindaria A,  Aust SD.  EPR detection and characterization of lignin peroxidase porphyrin π-cation radical,  Biochemistry ,  1996, vol.  35 (pg.  13107- 13111) Google Scholar CrossRef Search ADS PubMed  Khindaria A,  Nie G,  Aust SD.  Detection and characterization of the lignin peroxidase compound II–veratryl alcohol cation radical complex,  Biochemistry ,  1997, vol.  36 (pg.  14181- 14185) Google Scholar CrossRef Search ADS PubMed  Kirk TK,  Farrell RL.  Enzymatic ‘combustion’: the microbial degradation of lignin,  Annual Review of Microbiology ,  1987, vol.  41 (pg.  465- 505) Google Scholar CrossRef Search ADS PubMed  Kishi K,  Kusters-van Someren M,  Mayfield MB,  Sun J,  Loehr TM,  Gold MH.  Characterization of manganese(II) binding site mutants of manganese peroxidase,  Biochemistry ,  1996, vol.  35 (pg.  8986- 8994) Google Scholar CrossRef Search ADS PubMed  Koduri RS,  Tien M.  Oxidation of guaiacol by lignin peroxidase. Role of veratryl alcohol,  Journal of Biological Chemistry ,  1995, vol.  270 (pg.  22254- 22258) Google Scholar CrossRef Search ADS PubMed  Kusters-van Someren M,  Kishi K,  Lundell T,  Gold MH.  The manganese binding site of manganese peroxidase: characterization of an Asp179Asn site-directed mutant protein,  Biochemistry ,  1995, vol.  34 (pg.  10620- 10627) Google Scholar CrossRef Search ADS PubMed  Larrondo L,  Gonzalez A,  Perez AT,  Cullen D,  Vicuna R.  The nop gene from Phanerochaete chrysosporium encodes a peroxidase with novel structural features,  Biophysical Chemistry ,  2005, vol.  116 (pg.  167- 173) Google Scholar CrossRef Search ADS PubMed  Lisov AV,  Leontievsky AA,  Golovleva LA.  Hybrid Mn-peroxidases from basidiomycetes,  Applied Biochemistry and Microbiology ,  2007, vol.  43 (pg.  536- 543) Google Scholar CrossRef Search ADS   Lú-Chau TA,  Ruiz-Dueñas FJ,  Camarero S,  Feijoo G,  Martínez MJ,  Lema JM,  Martínez AT.  Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans,  Bioprocesses and Biosystems Engineering ,  2004, vol.  26 (pg.  287- 293) Google Scholar CrossRef Search ADS   Maeda Y,  Kajiwara S,  Ohtaguchi K.  Manganese peroxidase gene of the perennial mushroom Elfvingia applanata: cloning and evaluation of its relationship with lignin degradation,  Biotechnology Letters ,  2001, vol.  23 (pg.  103- 109) Google Scholar CrossRef Search ADS   Magliozzo RS,  Marcinkeviciene JA.  The role of Mn(II)-peroxidase activity of mycobacterial catalase-peroxidase in activation of the antibiotic isoniazid,  Journal of Biological Chemistry ,  1997, vol.  272 (pg.  8867- 8870) Google Scholar CrossRef Search ADS PubMed  Marques G,  Gamelas JAF,  Evtuguin D,  Ruiz-Dueñas FJ,  Morales M,  del Río JC,  Martínez AT,  Gutiérrez A.  Versatile peroxidase reoxidation of Mn-based polyoxomethalate in chlorine-free bleaching of eucalypt pulp,  Proceedings of the 8th international peroxidase symposium. Tampere ,  2008  20–23 August Martínez AT.  Molecular biology and structure-function of lignin-degrading haem peroxidases,  Enzyme and Microbial Technology ,  2002, vol.  30 (pg.  425- 444) Google Scholar CrossRef Search ADS   Martínez AT.  Polaina J,  MacCabe AP.  High redox potential peroxidases,  Industrial enzymes: structure, function and applications ,  2007 Berlin Springer(pg.  475- 486) Martínez AT,  Speranza M,  Ruiz-Dueñas FJ,  Ferreira P,  Camarero S,  Guillén F,  Martínez MJ,  Gutiérrez A,  del Río JC.  Biodegradation of lignocellulosics: microbiological, chemical and enzymatic aspects of fungal attack to lignin,  International Microbiology ,  2005, vol.  8 (pg.  195- 204) Google Scholar PubMed  Martínez MJ,  Ruiz-Dueñas FJ,  Guillén F,  Martínez AT.  Purification and catalytic properties of two manganese-peroxidase isoenzymes from Pleurotus eryngii,  European Journal of Biochemistry ,  1996, vol.  237 (pg.  424- 432) Google Scholar CrossRef Search ADS PubMed  Matsubara M,  Suzuki J,  Deguchi T,  Miura M,  Kitaoka Y.  Characterization of manganese peroxidases from the hyperligninolytic fungus IZU-154,  Applied and Environmental Microbiology ,  1996, vol.  62 (pg.  4066- 4072) Google Scholar PubMed  Mester T,  Ambert-Balay K,  Ciofi-Baffoni S,  Banci L,  Jones AD,  Tien M.  Oxidation of a tetrameric nonphenolic lignin model compound by lignin peroxidase,  Journal of Biological Chemistry ,  2001, vol.  276 (pg.  22985- 22990) Google Scholar CrossRef Search ADS PubMed  Mester T,  Tien M.  Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium,  Biochemical and Biophysical Research Communications ,  2001, vol.  284 (pg.  723- 728) Google Scholar CrossRef Search ADS PubMed  Miki Y,  Tanaka H,  Nakamura M,  Wariishi H.  Isolation and characterization of a novel lignin peroxidase from the white-rot basidiomycete Trametes cervina,  Journal of the Faculty of Agriculture of Kyushu University ,  2006, vol.  51 (pg.  99- 104) Miyazaki C,  Takahashi H.  Engineering of the H2O2-binding pocket region of a recombinant manganese peroxidase to be resistant to H2O2,  FEBS Letters ,  2001, vol.  509 (pg.  111- 114) Google Scholar CrossRef Search ADS PubMed  Miyazaki-Imamura C,  Oohira K,  Kitagawa R,  Nakano H,  Yamane T,  Takahashi H.  Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high-throughput screening using in vitro expression with protein disulfide isomerase,  Protein Engineering ,  2003, vol.  16 (pg.  423- 428) Google Scholar CrossRef Search ADS PubMed  Moreira PR,  Almeida-Vara E,  Malcata FX,  Duarte JC.  Lignin transformation by a versatile peroxidase from a novel Bjerkandera sp strain,  International Biodeterioration and Biodegradation ,  2007, vol.  59 (pg.  234- 238) Google Scholar CrossRef Search ADS   Moreira PR,  Duez C,  Dehareng D,  Antunes A,  Almeida-Vara E,  Frère JM,  Malcata FX,  Duarte JC.  Molecular characterisation of a versatile peroxidase from a Bjerkandera strain,  Journal of Biotechnology ,  2005, vol.  118 (pg.  339- 352) Google Scholar CrossRef Search ADS PubMed  Morgenstern I,  Klopman S,  Hibbett DS.  Molecular evolution and diversity of lignin degrading haem peroxidases in the agaricomycetes,  Journal of Molecular Evolution ,  2008, vol.  66 (pg.  243- 257) Google Scholar CrossRef Search ADS PubMed  Paszczynski A,  Crawford RL.  Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol,  Biochemistry and Biophysics Research Communications ,  1991, vol.  178 (pg.  1056- 1063) Google Scholar CrossRef Search ADS   Pelletier H,  Kraut J.  Crystal structure of a complex between electron transfer partners, cytochrome c peroxidase and cytochrome c,  Science ,  1992, vol.  258 (pg.  1748- 1755) Google Scholar CrossRef Search ADS PubMed  Pérez-Boada M,  Doyle WA,  Ruiz-Dueñas FJ,  Martínez MJ,  Martínez AT,  Smith AT.  Expression of Pleurotus eryngii versatile peroxidase in Escherichia coli and optimisation of in vitro folding,  Enzyme and Microbial Technology ,  2002, vol.  30 (pg.  518- 524) Google Scholar CrossRef Search ADS   Pérez-Boada M,  Ruiz-Dueñas FJ,  Pogni R,  Basosi R,  Choinowski T,  Martínez MJ,  Piontek K,  Martínez AT.  Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigations of three long-range electron transfer pathways,  Journal of Molecular Biology ,  2005, vol.  354 (pg.  385- 402) Google Scholar CrossRef Search ADS PubMed  Pfister TD,  Mirarefi AY,  Gengenbach AJ, et al.  Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase,  Journal of Biological Inorganic Chemistry ,  2007, vol.  12 (pg.  126- 137) Google Scholar CrossRef Search ADS PubMed  Pogni R,  Baratto MC,  Giansanti S,  Teutloff C,  Verdin J,  Valderrama B,  Lendzian F,  Lubitz W,  Vázquez-Duhalt R,  Basosi R.  Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta,  Biochemistry ,  2005, vol.  44 (pg.  4267- 4274) Google Scholar CrossRef Search ADS PubMed  Pogni R,  Baratto MC,  Teutloff C,  Giansanti S,  Ruiz-Dueñas FJ,  Choinowski T,  Piontek K,  Martínez AT,  Lendzian F,  Basosi R.  A tryptophan neutral radical in the oxidized state of versatile peroxidase from Pleurotus eryngii: a combined multi-frequency EPR and DFT study,  Journal of Biological Chemistry ,  2006, vol.  281 (pg.  9517- 9526) Google Scholar CrossRef Search ADS PubMed  Pogni R,  Teutloff C,  Lendzian F,  Basosi R.  Tryptophan radicals as reaction intermediates in versatile peroxidases: multifrequency EPR, ENDOR and density functional theory studies,  Applied Magnetic Resonance ,  2007, vol.  31 (pg.  509- 526) Google Scholar CrossRef Search ADS   Reading NS,  Aust SD.  Engineering a disulfide bond in recombinant manganese peroxidase results in increased thermostability,  Biotechnology Progress ,  2000, vol.  16 (pg.  326- 333) Google Scholar CrossRef Search ADS PubMed  Rodakiewicz-Nowak J,  Jarosz-Wilkolazka A,  Luterek J.  Catalytic activity of versatile peroxidase from Bjerkandera fumosa in aqueous solutions of water-miscible organic solvents,  Applied Catalysis A: General ,  2006, vol.  308 (pg.  56- 61) Google Scholar CrossRef Search ADS   Rodríguez E,  Nuero O,  Guillén F,  Martínez AT,  Martínez MJ.  Degradation of phenolic and non-phenolic aromatic pollutants by four Pleurotus species: the role of laccase and versatile peroxidase,  Soil Biology and Biochemistry ,  2004, vol.  36 (pg.  909- 916) Google Scholar CrossRef Search ADS   Ruiz-Dueñas FJ,  Aguilar A,  Martínez MJ,  Zorn H,  Martínez AT.  Gene cloning, heterologous expression, in vitro reconstitution, and catalytic properties of a versatile peroxidase,  Biocatalysis and Biotransformation ,  2007, vol.  25 (pg.  276- 285) Google Scholar CrossRef Search ADS   Ruiz-Dueñas FJ,  Camarero S,  Pérez-Boada M,  Martínez MJ,  Martínez AT.  A new versatile peroxidase from Pleurotus,  Biochemistry Society Transactions ,  2001, vol.  29 (pg.  116- 122) Google Scholar CrossRef Search ADS   Ruiz-Dueñas FJ,  Martínez MJ,  Martínez AT.  Molecular characterization of a novel peroxidase isolated from the ligninolytic fungus Pleurotus eryngii,  Molecular Microbiology ,  1999, vol.  31 (pg.  223- 236) Google Scholar CrossRef Search ADS PubMed  Ruiz-Dueñas FJ,  Morales M,  Mate MJ,  Romero A,  Martínez MJ,  Smith AT,  Martínez AT.  Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase,  Biochemistry ,  2008, vol.  47 (pg.  1685- 1695) Google Scholar CrossRef Search ADS PubMed  Ruiz-Dueñas FJ,  Morales M,  Pérez-Boada M,  Choinowski T,  Martínez MJ,  Piontek K,  Martínez AT.  Manganese oxidation site in Pleurotus eryngii versatile peroxidase: a site-directed mutagenesis, kinetic and crystallographic study,  Biochemistry ,  2007, vol.  46 (pg.  66- 77) Google Scholar CrossRef Search ADS PubMed  Ruiz-Dueñas FJ,  Morales M,  Rencoret J,  Gutiérrez A,  del Río JC,  Martínez MJ,  Martínez AT. ,  Improved peroxidases. Patent (Spain) P200801292 ,  2008 Ruiz-Dueñas FJ,  Pogni R,  Morales M,  Martínez MJ,  Basosi R,  Martínez AT.  Tailoring alternative catalytic protein radicals in Pleurotus eryngii versatile peroxidase,  Proceedings of the 8th International Peroxidase Symposium. Tampere ,  2008  20–23 August Ryan BJ,  Carolan N,  O'Fagain C.  Horseradish and soybean peroxidases: comparable tools for alternative niches?,  Trends in Biotechnology ,  2006, vol.  24 (pg.  355- 363) Google Scholar CrossRef Search ADS PubMed  Ryu K,  Hwang SY,  Kim KH,  Kang JH,  Lee EK.  Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability,  Journal of Biotechnology ,  2008, vol.  133 (pg.  110- 115) Google Scholar CrossRef Search ADS PubMed  Schoemaker HE,  Lundell TK,  Floris R,  Glumoff T,  Winterhalter KH,  Piontek K.  Do carbohydrates play a role in lignin peroxidase cycle? Redox catalysis in the endergonic region of the driving force,  Bioorganic and Medicinal Chemistry ,  1994, vol.  2 (pg.  509- 519) Google Scholar CrossRef Search ADS PubMed  Schoemaker HE,  Lundell TK,  Hatakka AI,  Piontek K.  The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: the redox cycle revisited,  FEMS Microbiology Reviews ,  1994, vol.  13 (pg.  321- 332) Google Scholar CrossRef Search ADS   Sharp KH,  Mewies M,  Moody PC,  Raven EL.  Crystal structure of the ascorbate peroxidase–ascorbate complex,  Nature Structural Biology ,  2003, vol.  10 (pg.  303- 307) Google Scholar CrossRef Search ADS PubMed  Sheng D,  Gold MH.  Irreversible oxidation of ferricytochrome c by lignin peroxidase,  Biochemistry ,  1998, vol.  37 (pg.  2029- 2036) Google Scholar CrossRef Search ADS PubMed  Sigoillot C,  Camarero S,  Vidal T, et al.  Comparison of different fungal enzymes for bleaching high-quality paper pulps,  Journal of Biotechnology ,  2005, vol.  115 (pg.  333- 343) Google Scholar CrossRef Search ADS PubMed  Smith AT,  Doyle WA.  Engineered peroxidases with veratryl alcohol oxidase activity,  Patent (International) WO/2006–114616 ,  2006 Smith AT,  Veitch NC.  Substrate binding and catalysis in haem peroxidases,  Current Opinion in Chemical Biology ,  1998, vol.  2 (pg.  269- 278) Google Scholar CrossRef Search ADS PubMed  Sundaramoorthy M,  Kishi K,  Gold MH,  Poulos TL.  The crystal structure of manganese peroxidase from Phanerochaete chrysosporium at 2.06 Å resolution,  Journal of Biological Chemistry ,  1994, vol.  269 (pg.  32759- 32767) Google Scholar PubMed  Sundaramoorthy M,  Kishi K,  Gold MH,  Poulos TL.  Crystal structures of substrate binding site mutants of manganese peroxidase,  Journal of Biological Chemistry ,  1997, vol.  272 (pg.  17574- 17580) Google Scholar CrossRef Search ADS PubMed  Sundaramoorthy M,  Youngs HL,  Gold MH,  Poulos TL.  High-resolution crystal structure of manganese peroxidase: substrate and inhibitor complexes,  Biochemistry ,  2005, vol.  44 (pg.  6463- 6470) Google Scholar CrossRef Search ADS PubMed  Tamura K,  Dudley J,  Nei M,  Kumar S.  MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0,  Molecular Biology and Evolution ,  2007, vol.  24 (pg.  1596- 1599) Google Scholar CrossRef Search ADS PubMed  Timofeevski SL,  Nie G,  Reading NS,  Aust SD.  Engineering a functional hybrid of manganese peroxidase and lignin peroxidase,  Plant Peroxidase Newsletter ,  1999, vol.  13 (pg.  99- 111) Tinoco R,  Verdín J,  Vázquez-Duhalt R.  Role of oxidizing mediators and tryptophan 172 in the decoloration of industrial dyes by the versatile peroxidase from Bjerkandera adusta,  Journal of Molecular Catalysis B: Enzymatic ,  2007, vol.  46 (pg.  1- 7) Google Scholar CrossRef Search ADS   Tsukamoto K,  Itakura H,  Sato K,  Fukuyama K,  Miura S,  Takahashi S,  Ikezawa H,  Hosoya T.  Binding of salicylhydroxamic acid and several aromatic donor molecules to Arthromyces ramosus peroxidase, investigated by X-ray crystallography, optical difference spectroscopy, NMR relaxation, molecular dynamics, and kinetics,  Biochemistry ,  1999, vol.  38 (pg.  12558- 12568) Google Scholar CrossRef Search ADS PubMed  Tsukihara T,  Honda Y,  Sakai R,  Watanabe T,  Watanabe T.  Mechanism for oxidation of high-molecular-weight substrates by a fungal versatile peroxidase, MnP2,  Applied and Environmental Microbiology ,  2008, vol.  74 (pg.  2873- 2881) Google Scholar CrossRef Search ADS PubMed  Valderrama B,  Ayala M,  Vázquez-Duhalt R.  Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes,  Chemistry and Biology ,  2002, vol.  9 (pg.  555- 565) Google Scholar CrossRef Search ADS PubMed  Veitch NC.  Horseradish peroxidase: a modern view of a classic enzyme,  Phytochemistry ,  2004, vol.  65 (pg.  249- 259) Google Scholar CrossRef Search ADS PubMed  Viikari L,  Kantelinen A,  Sundquist J,  Linko M.  Xylanases in bleaching: from an idea to the industry,  FEMS Microbiology Reviews ,  1994, vol.  13 (pg.  335- 350) Google Scholar CrossRef Search ADS   Wang YX,  Vázquez-Duhalt R,  Pickard MA.  Manganese–lignin peroxidase hybrid from Bjerkandera adusta oxidizes polycyclic aromatic hydrocarbons more actively in the absence of manganese,  Canadian Journal of Microbiology ,  2003, vol.  49 (pg.  675- 682) Google Scholar CrossRef Search ADS PubMed  Welinder KG.  Superfamily of plant, fungal and bacterial peroxidases,  Current Opinion in Structural Biology ,  1992, vol.  2 (pg.  388- 393) Google Scholar CrossRef Search ADS   Whitwam RE,  Brown KR,  Musick M,  Natan MJ,  Tien M.  Mutagenesis of the Mn2+-binding site of manganese peroxidase affects oxidation of Mn2+ by both compound I and compound II.,  Biochemistry ,  1997, vol.  36 (pg.  9766- 9773) Google Scholar CrossRef Search ADS PubMed  Wilcox SK,  Putnam CD,  Sastry M,  Blankenship J,  Chazin WJ,  McRee DE,  Goodin DB.  Rational design of a functional metalloenzyme: Introduction of a site for manganese binding and oxidation into a haem peroxidase,  Biochemistry ,  1998, vol.  37 (pg.  16853- 16862) Google Scholar CrossRef Search ADS PubMed  Yeung BKS,  Wang XT,  Sigman JA,  Petillo PA,  Lu Y.  Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase,  Chemistry and Biology ,  1997, vol.  4 (pg.  215- 221) Google Scholar CrossRef Search ADS PubMed  Youngs HL,  Gelpke MDS,  Li DM,  Sundaramoorthy M,  Gold MH.  The role of Glu39 in Mn-II binding and oxidation by manganese peroxidase from Phanerochaete chrysosporium,  Biochemistry ,  2001, vol.  40 (pg.  2243- 2250) Google Scholar CrossRef Search ADS PubMed  © The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Journal

Journal of Experimental Botany – Oxford University Press

Published: Nov 5, 2008

Keywords: Crystal structures fungal peroxidases haem access-channel lignin biodegradation manganese-binding site site-directed mutagenesis spectroscopic analyses transient-state kinetics tryptophanyl radical versatile peroxidase

There are no references for this article.