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Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase *

Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 40, pp. 34316 –34323, October 7, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase- Methenyltetrahydrofolate Cyclohydrolase Received for publication, May 11, 2005, and in revised form, August 3, 2005 Published, JBC Papers in Press, August 11, 2005, DOI 10.1074/jbc.M505210200 ‡1 ‡2 ‡§3 ‡4 Karen E. Christensen , I. Ahmad Mirza , Albert M. Berghuis , and Robert E. MacKenzie ‡ § From the Department of Biochemistry and the Department of Microbiology and Immunology, McGill University, Montre´al, Que´bec H3G 1Y6, Canada The mitochondrial NAD-dependent methylenetetrahydrofolate dria. The mitochondrial formyl-THF is converted to formate by a mono- dehydrogenase-cyclohydrolase (NMDMC) is believed to have functional formyl-THF synthetase and is released to the cytoplasm for re- evolved from a trifunctional NADP-dependent methylenetetrahy- conversion into formyl-THF to support purine biosynthesis (1). NMDMC drofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in can use both NAD and NADP as a cofactor in its dehydrogenase activity, its absolute requirement for inorganic phosphate and magnesium although the maximal activity with NADP is only about twenty percent of ions to support dehydrogenase activity. To enable us to investigate that with NAD (3). NMDMC is thought to have evolved from a trifunc- the roles of these ions, a homology model of human NMDMC was tional NADP-dependent methylene-THF dehydrogenase-methenyl-THF constructed based on the structures of three homologous proteins. cyclohydrolase-formyl-THF synthetase (DCS) through the loss of the syn- The model supports the hypothesis that the absolutely required P thetase domain and the change in cofactor specificity from NADP to NAD can bind in close proximity to the 2-hydroxyl of NAD through (4). This change in cofactor specificity is important because the use of NAD 166 198 interactions with Arg and Arg . The characterization of rather than NADP in mitochondria shifts the equilibrium of the reaction to 166 190 198 166 mutants of Arg ,Asp , and Arg show that Arg is primarily favor the production of formyl-THF (5). The increased production of responsible for P binding, while Arg plays a secondary role, formyl-THF is required to meet the demand for glycine and purines during assisting in binding and properly orienting the ion in the cofactor embryogenesis (1, 2, 6). However, it is not known how this cofactor speci- 190 166 binding site. Asp helps to properly position Arg . Mutants of ficity change was accomplished. Asp suggest that the magnesium ion interacts with both P and NMDMC is unique in its absolute requirement for magnesium and the aspartate side chain and plays a role in positioning P and NAD. inorganic phosphate ions for NAD-dependent dehydrogenase activity NMDMC uses P and magnesium to adapt an NADP binding site for and magnesium ions for NADP-dependent dehydrogenase activity (3, NAD binding. This adaptation represents a novel variation of the 7). However, neither ion is essential for the cyclohydrolase activity. The classic Rossmann fold. role of these ions in the dehydrogenase activity is not clear. The sequence of binding of the cofactors and substrates to NMDMC, as established kinetically by Yang and Mackenzie (3) and Rios-Orlandi and During embryogenesis and tumorigenesis mammalian mitochondria MacKenzie (7), suggests a role for P and Mg in the binding of the use a folate-dependent pathway to generate both glycine and one-carbon cofactor; the ions bind to the protein first, followed by NAD and then units to support cytoplasmic purine synthesis (1, 2). One of the enzymes in the folate substrate. A preferred order of binding of the ions was not this pathway, the NAD-dependent methylenetetrahydrofolate dehydro- 5 established; either ion appears to be able to bind to the enzyme and genase-methenyltetrahydrofolate cyclohydrolase (NMDMC), catalyzes affect the binding of the other. These results suggested a possible inter- the interconversion of 5,10-methylenetetrahydrofolate (methylene-THF) action between the two ions in the binding site. and 10-formyltetrahydrofolate (formyl-THF) in mammalian mitochon- The observation that P competitively inhibits the cofactor in NADP-de- pendent dehydrogenase assays of NMDMC led to the proposal that P may * This work was supported in part by Canadian Institutes of Health Research (CIHR) Grant occupy a position adjacent to the 2-hydroxyl of NAD, close to the space 29814. The costs of publication of this article were defrayed in part by the payment of that would be occupied by the 2-phosphate of NADP (3). Previous work on page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the DC domain of the human NADP-dependent DCS identified two resi- The atomic coordinates and structure factors (code 1ZN4) have been deposited in the Protein 173 197 dues (Arg and Ser ) as being important to the binding of NADP to the Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). enzyme through its 2-phosphate (8). Sequence alignments of mitochon- Supported by the CIHR, Fonds de Recherche en Sante´ du Quebe´c, and McGill 166 drial NAD-DCs with trifunctional NADP-DCSs suggest that Arg and University. Arg (numbered from the amino-terminal glutamate of the mature Supported by the International Center for Diffraction Data. Recipient of a Canada Research Chair in Structural Biology. enzyme) may interact with P (9 and Fig. 1). 4 i To whom correspondence should be addressed: Dept. of Biochemistry, McGill Univer- The crystal structure of the DC domain of the human NADP-dependent sity, McIntyre Medical Sciences Bldg., 3655 Promenade Sir William Osler, Montre´al, Que´bec H3G 1Y6, Canada. Tel.: 514-398-7270; Fax: 514-398-7384; E-mail: DCS has been determined both with bound NADP and with bound NADP [email protected]. and folate analogues (10, 11). The structure of the Escherichia coli NADP- The abbreviations used are: NMDMC, NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; methylene-THF, 5,10- dependent DC has been determined by x-ray crystallography in the absence methylenetetrahydrofolate; formyl-THF, 10-formyltetrahydrofolate; DCS, methyl- of bound substrates (12), and the structure of the Saccharomyces cerevisiae enetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase- formyltetrahydrofolate synthetase; DC, methylenetetrahydrofolate dehydrogenase- NAD-dependent dehydrogenase has been determined with and without methenyltetrahydrofolate cyclohydrolase; GR, glutathione reductase; DH, bound NAD (13). However, since no crystal structure of NMDMC has yet dehydrogenase; PDB, Protein Data Bank; MOPS, 4-morpholinepropanesulfonic acid; WT, wild type. been obtained, we constructed a homology model of the enzyme based on 34316 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 This is an Open Access article under the CC BY license. Magnesium and Phosphate Binding Sites of NMDMC FIGURE 1. Structural alignment of human NMDMC with the templates used to construct the homology model. The templates are the DC domain of the human NADP-depend- ent DCS crystallized with NADP and a folate analogue (PDB ID: 1DIB), the E. coli NADP-dependent DC (PDB ID: 1B0A), and the S. cerevisiae NAD-dependent dehydrogenase crystallized with NAD (PDB ID: 1EE9). A, alignment of the primary structures of NMDMC and the templates adjusted to reflect the fit of the crystal structures 1B0A and 1EE9 to 1DIB. Residues that are aligned in the sequence are considered structurally homologous.-Helices are marked in red and-sheets in blue. Residues targeted in mutagenesis experiments are marked with an asterisk. B and C, overlay of the NMDMC model and the template structures, showing one monomer of the dimer from two sides. The 1B0A structure is cyan, the 1DIB structure is magenta, the 1EE9 structure is blue, and the NMDMC model is green. three related structures and used this to locate the P and Mg binding sites Construction of the Homology Model of Human NMDMC—Crystal using site-directed mutagenesis. structures for three NMDMC homologues have been determined. The DC domain of the human NADP-dependent DCS structure has been EXPERIMENTAL PROCEDURES solved with NADP (10) and with NADP and three different folate ana- Materials—(R,S)-tetrahydrofolate was synthesized according to the logues (11). The E. coli NADP-dependent DC structure has been solved method of Drury et al. (14) and stored in sealed glass vials at 4 °C. in the absence of ligands (12). The S. cerevisiae NAD-dependent dehy- Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen. Vent drogenase has been solved both without ligands and in complex with DNA polymerase was obtained from New England Biolabs, and restric- NAD (13). Templates with bound cofactors and substrates were tion enzymes were products of New England Biolabs and MBI Fermen- selected to build a holoenzyme model of human NMDMC. The three tas. Oligonucleotide primers were obtained from Sigma Genosys, Qia- template structures were obtained from the Protein Data Bank (PDB): gen Operon, and Integrated DNA Technologies, Inc. NAD, imidazole, the DC domain of the human NADP-dependent DCS in complex with and rifampicin were purchased from Sigma. (R,S)-methenyltetrahydro- NADP and a folate analogue (PDB ID: 1DIB), the E. coli NADP-depend- folate was obtained from B. Schircks Laboratories (Jona, Switzerland). ent DC (PDB ID: 1B0A), and the S. cerevisiae NAD-dependent dehydro- All other chemicals and reagents were purchased from Bioshop or BDH genase in complex with NAD (PDB ID: 1EE9). Although the sequence and were of analytical grade. similarity among these proteins is not particularly high as the pair-wise OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34317 Magnesium and Phosphate Binding Sites of NMDMC TABLE ONE Dehydrogenase activity of Arg mutants Specific activity (% WT) Enzyme NAD-DH NADP-DH 1 1 mol min mg WT 22.5  1.1 (100) 2.92  0.06 (100) R166A ND ND R166S ND ND R166K ND ND a 1 1 ND  not detectable (0.01 mol min mg ). TABLE TWO Dehydrogenase activity of Asp mutants Specific activity (% WT) Enzyme NAD-DH NADP-DH 1 1 mol min mg WT 22.5  1.1 (100) 2.92  0.06 (100) D190A ND ND D190S ND ND D190E 0.024  0.001(0.1) ND FIGURE 2. Ribbon diagram of NMDMC homology model with docked NAD and inor- ganic phosphate. D190N 3.64  0.03 (16) 3.96  0.07 (136) a 1 1 ND  not detectable (0.01 mol min mg ). optimization residues 243 and 253 were fixed to keep their position homologous to the 1DIB structure. NAD, P , and NADP were docked into the model based on the position of the cofactors in the template structures. The crystal structures 1B0A and 1EE9 show only one mon- omer of the enzymes and so the resulting model showed only one mon- omer of NMDMC, which is known to be a dimer (21). To model the dimer two copies of the model were superimposed on the 1DIB struc- ture using Lsqman (16), and the geometry and stereochemistry of the side chains along the dimer interface was optimized using CNS (19). The quality of the model was evaluated at every step using Procheck (22). Pictures of the model were generated using Pymol (23). FIGURE 3. Comparison of the cofactor binding sites of NMDMC and the DC domain Addition of Six-histidine Tags—To simplify purification a COOH- of the human DCS. A, the NAD and inorganic phosphate binding site of the NMDMC 166 198 133 190 model. Arg and Arg are found in monomer A and Asp and Asp in monomer B. terminal six-histidine tag was added to NMDMC by PCR using primers B, the NADP binding site of the 1DIB structure. including the 3 terminus of the required coding region of the cDNA, the sequence of the tag and an XhoI site. The histidine-tagged cDNA percent identity ranges between 22–43%, the three-dimensional struc- was then subcloned into pBKeHB1 (24) to make pBKeHB1 303H6. The tures are highly homologous (see Fig. 1). six-histidine tag replaces an unstructured tail made up of twelve resi- A multiple sequence alignment of the target and template sequences dues that have no influence on enzyme activity. The kinetic constants was generated using ClustalW (15). The alignment was then refined to and activities of 303H6 are identical to the full-length, non-histidine- include structural data using alignments to the 1DIB structure gener- tagged NMDMC (data not shown), and so the histidine tag does not ated using a brute force structural alignment method followed by iter- interfere with the enzyme function. The truncated version of the pro- ative cycles of improvement as implemented in the program Lsqman tein was selected for this study because it had better expression levels (16). Structural elements for the model were selected from the tem- than full-length NMDMC with six histidines added and to circumvent plates by highest sequence identity to NMDMC. The structural ele- any problems that may arise from having the six-histidine tag added to ments from the aligned templates were linked together and the side the mobile unstructured tail. For simplicity, in the results section 303H6 chains mutated using the computer program O (17) with the Lovell et al. is referred to as wild type, meaning unmutated pBKeHB1 303H6. (18) rotamer data base. Site-directed Mutagenesis—Mutations were introduced into The loop from residue 182 to 190, unique to mitochondrial NAD-de- pBKeHB1 303H6 using in vitro overlap extension PCR as in Sundarara- pendent DCs (9 and Fig. 1), lacks structural data and was not included in jan and MacKenzie (20). The entire insert of the resulting vector was the final structure. The position of Asp suggested that it could play a sequenced by automated sequencing (Genome Que´bec) to confirm the role in the cofactor binding site, so it was included in the model, and its integrity of each mutant. position was determined by local energy minimization using CNS (19). All of the mutants in this study should affect the NAD(P) cofactor The loop from residue 244 to 252 is disordered in the 1DIB structure binding and thus only the dehydrogenase activity, leaving the cyclohy- and is not present in the E. coli and S. cerevisiae enzymes. This loop has drolase activity and folate substrate binding kinetic constants as con- been shown not to be required for DH activity or cofactor binding (20) trols for gross disruptions of the protein structure. All of the mutants and so was not included in the model. The geometry and stereochem- reported in this study retained significant cyclohydrolase activity. There istry of the model structure was optimized using CNS (19). For the were no significant differences between the K values for methylene- 34318 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 Magnesium and Phosphate Binding Sites of NMDMC TABLE THREE Kinetic constants of D190N compared to wild-type Phosphate Mg (NAD) K Enzyme K of NADP-DH K K n NAD Mg (NADP) NADP i m mM M M M WT 4.13 412  55 351  21 1.99  0.10 202  13 321  36 352  41 D190N 3.43 3100  570 2050  130 1.62  0.11 3880  620 1990  460 689  121 THF, with either NAD or NADP as the cofactor, or for methenyl-THF TABLE FOUR for any of the mutants. This shows that there are no gross disruptions of Dehydrogenase activity of Arg mutants the protein structure in the mutants and that the folate substrate bind- Specific activity (% WT) ing site is unaffected by these mutations. Enzyme NAD-DH NADP-DH Protein Expression and Purification—pBKeHB1 303H6 constructs 1 1 mol min mg expressing histidine-tagged NMDMC were transformed into E. coli WT 22.5  1.1 (100) 2.92  0.06 (100) BL21 DE3. Overnight cultures of transformed bacteria were used to R198A 0.170  0.032 (0.8) 0.156  0.021 (5.3) inoculate 100 ml of Terrific Broth supplemented with 200 g/ml ampi- R198S 0.207  0.018 (0.9) 1.54  0.09 (53) cillin and incubated at 37 °C with shaking at 250 rpm. Expression was R198K 12.6  1.2 (56) 1.24  0.05 (42) induced when cultures reached an A 1.0 to 1.2 by adding isopropyl 1-thio--D-galactopyranoside to a final concentration of 2 mM. After 30 Schmidt et al. (11), typically using four time points at 2-min intervals min 15 g/ml rifampicin was added to inhibit host translation. Cells and at least five variable substrate concentrations. were harvested after an additional 90 min by centrifugation for 20 min at Cyclohydrolase assays were performed as described in Pawelek and 4000 rpm at 4 °C in a Sorvall RC-3. Pellets of 0.3 to 0.4 g cells were MacKenzie (26), with 5 mM magnesium chloride added to the buffer. stored at 85 °C. Results reported are the average of three separate determinations done Frozen pellets were thawed on ice and resuspended in 10 ml of son- in triplicate. The determination of kinetic constants was done as for the ication buffer (0.1 M potassium phosphate (pH 7.3), 35 mM -mercap- standard cyclohydrolase assay, using at least five methenyl-THF toethanol, 1 mM benzamidine hydrochloride, and 1 mM phenylmethyl- concentrations. sulfonyl fluoride). Resuspended cells were disrupted by sonication on For enzymes with sufficient activity, initial rate data were fitted to the ice using 10–12 pulses of 10 s each separated by intervals of 1 min. Michealis-Menten equation by non-linear regression using Sigmaplot Lysates were cleared by centrifugation at 12,500 rpm at 4 °C in a Sorvall (Systat Software Inc.). The kinetic constants for Mg with NAD as a SS-34 rotor for 20 min. Protamine sulfate (0.1 volume of 10 mg/ml cofactor were determined in the same manner by fitting to the Hill solution) was added to the supernatant, followed by an additional equation. Standard errors of the fit for K and V were under 25%. 20-min centrifugation at 12,500 rpm. m max Results are reported as the average and standard deviation of three to The protamine sulfate-treated crude extract was adjusted to contain five separate K determinations. 0.5 M NaCl and 15 mM imidazole and added to 10 ml of a 50% slurry of m Inhibition of NADP-dehydrogenase activity by P was examined by Ni-NTA-agarose resin in binding buffer (0.1 M potassium phosphate performing assays at four fixed P concentrations at varying concentra- (pH 7.8), 0.5 M NaCl, 15 mM imidazole, 1 mM phenylmethylsulfonyl i tions of NADP, similar to Yang and MacKenzie (3). K values for P fluoride, 1 mM benzamidine, 10 mM -mercaptoethanol, and 20% (v/v) i i against NADP were calculated from the intercepts of replots of the glycerol). The slurry was mixed for 1.5 h at 4 °C on a rotator, and the slopes of Lineweaver-Burk plots versus P concentration. resin was collected by centrifuging at 1,000 rpm for 10 min in a Sorvall RC-3. The resin was then resuspended in 5 ml of binding buffer and RESULTS packed into a 1.5-cm diameter column. The column was washed once at NMDMC Homology Model 0.25 ml/min with 3 volumes of binding buffer, followed by 3 volumes of binding buffer containing 50 mM imidazole. The enzyme was eluted at The homology model of NMDMC is in good agreement with the 0.1 ml/min with binding buffer containing 250 mM imidazole. Fractions template structures. The root mean square deviation of the  carbons containing the enzyme were identified by Bradford assay (25). All between the model and each template is as follows: 1DIB structure enzyme preparations were evaluated for purity by SDS-PAGE on 10% (human DCS), 1.23 Å; 1B0A structure (E. coli DC), 0.92 Å; and 1EE9 polyacrylamide gels. Protein concentration was determined by Bradford structure (S. cerevisiae D), 1.40 Å (see Fig. 1). The Ramachandran plot of assay performed in triplicate, using bovine serum albumin as a standard. the model demonstrates that 82.6% of residues are in core regions, 14.6% Enzyme Assays and Kinetics—Dehydrogenase assays were performed in allowed regions, and 2.8% in generously allowed regions. No residues after Yang and Mackenzie (3) and Pawelek and MacKenzie (26). Stand- are found in disallowed conformations. A ribbon diagram of the homol- ard conditions buffer contained 25 mM MOPS (pH 7.3), 5 mM potassium ogy model, shown with docked P and NAD, is shown in Fig. 2. The phosphate (pH 7.3), 5 mM magnesium chloride, 2.5 mM formaldehyde, coordinates for the model have been deposited in the Protein Data Bank 0.2 mM (6R,S)-tetrahydrofolate, 36 mM -mercaptoethanol, and 0.6 mM with accession number 1ZN4. NAD. For NADP-dependent DH assays the assay mixture was modified The Phosphate Binding Site of NMDMC by removing the potassium phosphate and NAD and including 2 mM NADP. Standard activity assays are reported as the average of three The competitive inhibition of P against NADP in dehydrogenase separate determinations performed in triplicate using a single fixed time assays lead Yang and MacKenzie (3) to propose that P binds to point. The determination of kinetic constants was performed on NMDMC in a position analogous to the location of the 2-phosphate of mutants with sufficient activity using a multiple time point assay as in NADP in DCS. The NAD binding site of the NMDMC model is shown OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34319 Magnesium and Phosphate Binding Sites of NMDMC TABLE FIVE Kinetic constants of Arg mutants compared to wild-type Phosphate Mg (NAD) K Enzyme K of NADP-DH K K n NAD Mg (NADP) NADP i m mM M M M WT 4.13 412  55 351  21 1.99  0.10 202  13 321  36 352  41 R198S 10.2 ND ND ND ND 349  49 255  47 R198K 9.35 2230  530 1470  100 1.49  0.08 918  190 1110  80 2120  20 ND  not determined due to insufficient enzyme activity. 190 166 residue that disrupted the ionic interaction of Asp and Arg (such TABLE SIX as D190A and D190S) or resulted in a shift of the position of Arg in Dehydrogenase activity of Asp mutants the binding site (D190E) resulted in the loss of DH activity. Only D190N Specific activity (% WT) retained significant amounts of DH activity with both cofactors. This Enzyme NAD-DH NADP-DH mutant would have a weaker interaction between residues 166 and 190, 1 1 mol min mg 166 166 allowing greater movement of Arg , but the position of Arg would WT 22.5  1.1 (100) 2.92  0.06 (100) not shift due to the similar size of the Asp and Asn side chains. The D133A 0.392  0.037 (2) 0.160  0.028 (6) affinity of D190N for P , as measured by its inhibition constant against D133S 3.99  0.33 (18) 1.01  0.06 (35) NADP, is not changed although all values of K for substrates and ions D133E ND ND are elevated. These changes in the K values most likely reflect the D133N 4.59  0.24 (20) 2.40  0.15 (82) inhibition of DH activity due to the difficulty of positioning the ligands a 1 1 ND  not detectable (0.01 mol min mg ). properly in the binding site when Arg , and therefore P , are free to 2 2 move. The increase in the Mg constants suggests that the Mg and P compared with the NADP binding site of DCS in Fig. 3. The P modeled binding sites, like the NAD and P binding sites, interact with each other. in the NMDMC structure is slightly displaced from NAD into a posi- The K for NADP is less affected than that for NAD, because the 166 198 tively charged cavity made up of Arg and Arg . Three of the P i 2-phosphate is covalently attached and requires positioning of a single oxygens are now within hydrogen bonding distance (2.5–3.5 Å) of the ligand rather than two separate molecules. Binding NADP might help to 166 198 2- and 3-hydroxyls of the NAD ribose. Arg and Arg are homol- partially reposition Arg in the D190N mutant. These mutations show 173 197 190 166 ogous to Arg and Ser , respectively, of the human DCS, which are that the role of Asp is to position Arg in the binding site. 198 198 involved in NADP binding (8). Mutagenesis of Arg —The properties of Arg mutants are shown Arg of DCS forms multiple hydrogen bonds with and stabilizes the in TABLES FOUR and FIVE. In the case of R198A, where the arginine is charge of the 2-phosphate of NADP (10). When this residue is mutated, substituted with a residue that is not capable of forming hydrogen dehydrogenase activity of DCS is reduced to less than 2.5% of wild-type bonds, both NAD-DH and NADP-DH activities are significantly activity, and values of K for NADP are greatly increased, showing the reduced, indicating an important role for this residue (TABLE FOUR). importance of this residue in binding the cofactor (8). In the homology When this arginine is substituted by serine, which is the homologous model (Fig. 3) Arg appears to have the potential to form multiple residue in DCS, NAD-DH activity is drastically reduced, while the hydrogen bonding interactions with P and can also contribute to stabi- NADP-DH is relatively unaffected. The P affinity, as measured by the K 173 i i lizing the charge of P , similar to the role of Arg in DCS. Therefore this for P , is reduced in this mutant, which shows that Arg does assist in residue was targeted for mutagenesis. P binding. The K values for Mg and NADP are not affected by this 197 i m Ser of the human DCS forms one hydrogen bond with the 2-phos- mutation, which shows that the binding site of Mg is not distorted in phate of NADP (10) and was shown by mutagenesis to play a supporting 166 2 R198S and that the combination of Arg and Mg is sufficient to role in cofactor binding (8). In NMDMC (Fig. 3) Arg is the homolo- allow wild-type affinity for NADP. gous residue to Ser of DCS. In the homology model of NMDMC it The R198K mutant, in which the arginine is replaced with a smaller appears that this residue has the potential to form hydrogen bonds with and more flexible residue that also has a positive charge, retains roughly P and can also contribute to charge stabilization and so it too was 50% of both NAD and NADP-dependent DH activities. In R198K all of targeted for mutagenesis. 190 the K values are significantly increased compared with wild type. The Upon examination of the model, the side chain of Asp , a residue 166 K values in this case likely reflect both the loss of affinity for P (shown m i unique to NMDMC, was observed behind Arg in a position that by the larger K value for P ), and the difficulty of properly positioning i i suggests an electrostatic interaction (Fig. 3). Mutation of this residue the P in the binding site due to the increased flexibility of the side chain. provides the opportunity to manipulate the position of Arg in the The Mg constants, as in D190N, are also affected by this mutation, protein and observe the effects, without actually changing the nature of again showing that the two binding sites interact with each other. In this the side chain. 166 166 case, in contrast to R198S, the bulky side chain may be occluding the Mutagenesis of Arg —Even conservative mutations of Arg abro- Mg site or distorting it in a way that impairs binding. gate dehydrogenase activity (TABLE ONE). This is consistent with the 173 173 The role of Arg is illustrated by the effects of mutations on the K role of Arg in DCS. Arg is primarily responsible for NADP binding value for P . Although R198S and R198K have roughly the same affinity through the 2-phosphate, which suggests that Arg has a similar role i for Pi, as measured by the K , R198S almost totally lacks NAD-DH activ- in binding P in NMDMC. Mutagenesis of Asp —To examine the effects of changing the posi- ity, whereas R198K retains greater than 50%. Simply binding the phos- 166 190 tion of Arg without altering the side chain Asp was mutated. The phate is insufficient suggesting it must also be positioned properly for assay results are shown in TABLES TWO and THREE. Mutations of this DH activity by an electrostatic interaction with a positive charge. Arg 34320 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 Magnesium and Phosphate Binding Sites of NMDMC TABLE SEVEN Kinetic constants of Asp mutants compared to wild-type Phosphate Mg (NAD) K Enzyme K of NADP-DH K K n NAD Mg (NADP) NADP i m mM M M M WT 4.13 412  55 351  21 1.99  0.10 202  13 321  36 352  41 D133S 2.60 1320  180 2780  420 1.83  0.55 2170  190 3440  570 503  43 D133N 2.95 1250  240 2450  80 1.22  0.00 1990  300 3680  570 580  40 positions the phosphate that interacts with Arg and orients it within the binding site to interact with NAD. The Magnesium Binding Site Kinetics with Magnesium—A more sensitive multiple time point assay was used to determine K values for this study, as opposed to the single time point assay used previously (3). This method revealed a previously unobserved co-operativity in the binding of Mg with NAD. The velocity versus [Mg ] curve with NAD as a cofactor is sigmoidal and fits to the Hill equation. The value of n for 303H6 was found to be 1.99 0.10 indicating near perfect co-operativity of binding. This effect is not observed in velocity versus [Mg ] curves using NADP as a cofac- tor nor when [P ] is the variable substrate. Consequently both K and n FIGURE 4. The magnesium ion binding site may be located in a cavity bounded by values are reported for magnesium. The significance of this apparent Asp , the inorganic phosphate, and the NAD cofactor, which could provide co- co-operativity is not clear. ordination points for the cation. Identification of Potential Magnesium Binding Sites—Potential Mg 166 198 binding sites were identified by examining a multiple sequence align- the human DCS. In NMDMC, Arg and Arg provide a positively ment of methylene-THF dehydrogenases to find aspartate and gluta- charged pocket for binding P . Arg is the residue that is primarily mate residues that are conserved in human, mouse, and fruit fly responsible for binding P ; all mutants of this residue, even the conserv- NMDMC but not conserved in NADP-dependent enzymes that do not ative mutation to lysine, lack dehydrogenase activity. The lack of activity require Mg . These residues in particular were selected because they of R166K is not that surprising given that arginine residues are often are the preferred interacting partners of Mg , at least one of them favored over lysine at P sites because they can form multiple interac- being found in all known Mg binding sites (27). This search narrowed tions with the ion and can be resonance stabilized (28, 29). down the potential interacting residues to 8 from 36. The local environ- Mutagenesis of Asp shows that this residue is required to properly ment of these residues was examined in the homology model to deter- position Arg in the binding site. This type of arginine-aspartate inter- mine whether any could contribute to a Mg binding site. The exist- action to position the side chain has been previously observed in other ence of a possible binding partner to permit charge balance, the size of proteins that bind phosphate ions (30, 31, 32). Mutation of Asp to the the potential site (5–6 Å distance between binding partners), the larger glutamate residue displaces Arg and essentially inactivates charge of the site, and its access to the solvent were evaluated. Of the NMDMC. The D190N mutation retains significant DH activity. How- eight residues, only four are situated such that they could be part of a ever, the weakened interaction with Arg allows the residue to move, Mg binding site. Preliminary mutagenesis experiments eliminated impeding ligand binding, which increases the ligand K constants. 133 166 three of these residues leaving Asp as the only residue likely to con- These mutations show that the position of Arg is critical for cofactor tribute to a Mg binding site. binding to NMDMC. 133 133 198 166 198 Mutagenesis of Asp —The properties of the Asp mutants are Arg Positions Phosphate in the Binding Site—Unlike Arg , Arg shown in TABLES SIX and SEVEN. Substitution of aspartate by gluta- can be mutated to other residues without losing all dehydrogenase mate completely inactivates the DH activity with both NAD and NADP activity. The dehydrogenase activities and kinetic constants of Arg cofactors, indicating that the position of the carboxyl group is critical. mutants with NAD and NADP show that this residue assists in P bind- However, D133A, D133S, and D133N, without free carboxyl groups, ing but is not essential for the binding of NADP. A comparison of the K retain some DH activity. The D133S and D133N mutants show no for P and dehydrogenase activities of R198S and R198K establishes that reduction in affinity for P , as indicated by the K values, but the values of the role of Arg is not only to assist in binding P to NMDMC but also i i i 2 133 K for Mg are greatly elevated. These results support a role for Asp to position P within the binding site to optimize the interactions that m i 2 2 198 190 in helping to bind Mg . The loss of Mg affinity in these mutants allow NAD to bind. The Arg and Asp mutants both suggest that affects the positioning of P in the binding site and results in elevated the position of the phosphate is critical when it is not covalently bound 166 198 kinetic constants for all the ligands. This suggests that the role of the to the dinucleotide cofactor. The mutagenesis of Arg and Arg , Mg ion is to assist in the binding and positioning of P , much like the which are homologous to the 2-phosphate binding residues of DCS, role of Arg . confirms the hypothesis of Yang and MacKenzie (3) that P binds to NMDMC near the 2-hydroxyl of NAD. P in this position can form DISCUSSION multiple hydrogen bonds with the NAD cofactor. The K values for The Position of Arg Is Critical for Phosphate Binding—The model NAD and NADP are similar in the wild-type enzyme, but the V for max 166 198 of NMDMC shows that residues Arg and Arg are homologous to the NADP activity is only 20% of that with NAD and P .WhenNADis 173 197 Arg and Ser that interact with the 2-phosphate of NADP bound to bound to NMDMC the hydrogen bond length between P , and the OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34321 Magnesium and Phosphate Binding Sites of NMDMC FIGURE 5. Comparison of the cofactor binding sites of alcohol dehydrogenase (PDB ID: 1HDX) (A), NMDMC (B), and the DC domain of the human DCS (C) (PDB ID: 1DIB). The diphosphate binding domain of NMDMC lacks the close contact of protein and cofactor that is seen in the classic NAD binding site of alcohol dehydrogenase. This domain is more similar to the DC domain of DCS, which is entirely dependent on interactions with the 2-phosphate of NADP for cofactor binding (8). The magnesium and inorganic phosphate ions in the cofactor binding site of NMDMC set up a web of ionic and hydrogen bonding interactions that compensate for the lack of the covalent bond with the phosphate. This allows the adaptation of an NADP binding site to bind NAD. ribose moiety is longer than the bond length of the 2-phosphate of been mutated by Scrutton et al. (36) to preferentially use NADH. In that NADP. Therefore, when bound to the P site of NMDMC, NADP will protein seven residues, including the arginines, were mutated to alter shift upwards in the binding site, moving the nicotinamide moiety the cofactor binding region to more strongly resemble a classic NADH slightly out of position, affecting the activity of the enzyme but not the binding site. affinity for NADP. The interaction of Mg and P with a ligand containing a sugar The Magnesium and Phosphate Ions Interact—Mutagenesis of the moiety is similar to the crystal structure of the rat 3-phosphoadenosine residues of the P binding site supports the interaction of the P and 5-phosphate/inositol 1,4-bisphosphate phosphatase with bound AMP, i i 2 2 2 Mg binding sites suggested earlier by the enzyme kinetics (3). Muta- P , and Mg ions (37). In this structure three Mg ions bind the P , i i tions such as D190N and R198K that alter the positioning of the P in the which forms hydrogen bonds with the 2- and 3-hydroxyls of the ribose binding site also result in reduced affinity for Mg and indicate an moiety. interaction of the binding sites. The R198S mutant, which has decreased The Structure of the Cofactor Binding Site Is Similar to That of NADP P affinity (elevated K ) without altering NADP binding, has no effect on Sites—When the cofactor binding site of NMDMC is compared with a i i Mg affinity. These results suggest that the phosphate ion itself might classic Rossmann NAD binding site and to the NADP binding site of make up part of the Mg binding site and suggests that the role of the DCS, the role for the ions becomes more apparent (Fig. 5). The classic Mg ion is to assist in the binding and positioning of P , much like the NAD site has multiple interactions between the cofactor and the protein role of Arg . to enhance cofactor binding. The GXGXXG consensus sequence inter- 2 133 AMg ion interacting with Asp could co-ordinate with P within acts with the pyrophosphate moiety and maintains close proximity to the cavity illustrated in Fig. 4. The Mg binding site of NMDMC is the cofactor to maximize interactions. In particular, the second glycine made up of Asp and P , and these negative charges provide the charge in this region is thought to be important for close contact because any balance for the ion. The remaining four co-ordination points of the side chain at this position would disrupt cofactor binding (35). NAD- Mg (27) are most likely provided by backbone carbonyl groups, the binding proteins also typically have a conserved aspartate residue that 3-hydroxyl of NAD, and water. These interactions place the Mg forms hydrogen bonds with the hydroxyl groups of the adenine ribose of within a box with corners at the carboxyl group of Asp , the P , the NAD (35, 38). In contrast, NADP binding sites depend on the interac- 166 2 carbonyl of Arg , and the 3-hydroxyl of NAD. The position of Mg tion of the 2-phosphate of NADP with an arginine side chain (38), as is suggests that the ion stabilizes the position of P and NAD in the binding the case with DCS (8, 10). The GXGXXG consensus sequence is not as site through hydrogen bonds and charge interactions. The P stabiliza- strictly conserved in NADP-binding proteins, and the aspartate residue tion role in NMDMC is similar to the role of Mg in many proteins that is no longer conserved. NMDMC and DCS share the consensus use ATP or other phosphorylated substrates or intermediates (33). sequence of GRSXXXG (residues 172–178 of DCS and 165–171 of Similarities to P and Mg Usage in GR and Rat 3-Phosphoad- NMDMC). The substitution of the second glycine by serine in this enosine 5-Phosphate/Inositol 1,4-Bisphosphate Phosphatase—The use region disrupts the close interactions usually required for NAD binding of P to help bind NAD in NMDMC is similar to NADH binding to (Fig. 5). NMDMC also lacks the conserved aspartate residue. Thus, the native glutathione reductase (GR; Ref. 34). GR preferentially uses cofactor binding site of NMDMC more closely resembles an NADP NADPH to reduce oxidized glutathione to glutathione. NADPH binds binding site than a classic NAD binding site. Mutagenesis experiments to GR through interactions with two arginines (218 and 224). GR can on DCS showed that NADP binding to the protein is almost entirely also use NADH as a cofactor; however, the affinity of GR for NADH is dependent on the interaction between the 2-phosphate and Arg (8); roughly 60-fold weaker than the affinity for NADPH, and it can only the other small interactions between the protein and the cofactor were bind to the protein in the presence of P (34). The P in GR binds roughly not sufficient for NADP binding. Given the similarity of the NMDMC i i in the same position as the 2-phosphate of NADPH, interacting with and DCS cofactor binding sites, it seems clear that the role of the ions in the two arginine side chains. Although the use of the two arginines is NMDMC is to compensate for the lack of a covalently bound phosphate common between GR and NMDMC, these motifs are not structurally group on the cofactor. The Mg and P ions mediate multiple hydrogen related; it is not possible to overlay these motifs in the structures. In GR bonding interactions that adapt an NADP site to bind NAD. the arginines are independent of the GXGXXG motif (residues 174– Several laboratories have attempted to engineer NADP-specific pro- 179) that interacts with the diphosphate moiety of NAD(P)H (35), teins to preferentially use NAD (36, 39, 40). These groups have used a whereas in NMDMC Arg is the second residue of the motif. GR has mutagenesis approach to alter multiple side chains around the cofactor 34322 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 Magnesium and Phosphate Binding Sites of NMDMC 16. Kleywegt, G. J. (1996) Acta Crystallogr. Sect. D Biol. Crystallogr. 52, 842–857 binding site to mimic the binding site of a homologous protein specific 17. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. for NAD. Nature has, through evolution, used an entirely different A 47, 110–119 approach to change the cofactor specificity of the mitochondrial meth- 18. Lovell, S. C., Word, J. M., Richardson, J. S., and Richardson, D. C. (2000) Proteins 40, ylene-THF dehydrogenase, producing a protein whose specificity for 389–408 19. Brunger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, NAD compares favorably to these engineered proteins (36, 39, 40). This R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., use of Mg and P to bind NAD to the active site of NMDMC repre- Simonson, T., Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, sents a novel variation of the Rossmann fold. 905–921 20. Sundararajan, S., and MacKenzie, R. E. (2002) J. Biol. Chem. 277, 18703–18709 Acknowledgments—We thank Peter Pawelek and Saravanan Sudararajan for 21. Mejia, N. R., Rios-Orlandi, E. M., and MacKenzie, R. E. (1986) J. Biol. Chem. 261, 9509–9513 helpful discussion. 22. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. 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(1994) Nucleic Acids Res. 22, 4673–4680 278, 49203–49214 OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34323 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase-Methenyltetrahydrofolate Cyclohydrolase *

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m505210200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 40, pp. 34316 –34323, October 7, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Magnesium and Phosphate Ions Enable NAD Binding to Methylenetetrahydrofolate Dehydrogenase- Methenyltetrahydrofolate Cyclohydrolase Received for publication, May 11, 2005, and in revised form, August 3, 2005 Published, JBC Papers in Press, August 11, 2005, DOI 10.1074/jbc.M505210200 ‡1 ‡2 ‡§3 ‡4 Karen E. Christensen , I. Ahmad Mirza , Albert M. Berghuis , and Robert E. MacKenzie ‡ § From the Department of Biochemistry and the Department of Microbiology and Immunology, McGill University, Montre´al, Que´bec H3G 1Y6, Canada The mitochondrial NAD-dependent methylenetetrahydrofolate dria. The mitochondrial formyl-THF is converted to formate by a mono- dehydrogenase-cyclohydrolase (NMDMC) is believed to have functional formyl-THF synthetase and is released to the cytoplasm for re- evolved from a trifunctional NADP-dependent methylenetetrahy- conversion into formyl-THF to support purine biosynthesis (1). NMDMC drofolate dehydrogenase-cyclohydrolase-synthetase. It is unique in can use both NAD and NADP as a cofactor in its dehydrogenase activity, its absolute requirement for inorganic phosphate and magnesium although the maximal activity with NADP is only about twenty percent of ions to support dehydrogenase activity. To enable us to investigate that with NAD (3). NMDMC is thought to have evolved from a trifunc- the roles of these ions, a homology model of human NMDMC was tional NADP-dependent methylene-THF dehydrogenase-methenyl-THF constructed based on the structures of three homologous proteins. cyclohydrolase-formyl-THF synthetase (DCS) through the loss of the syn- The model supports the hypothesis that the absolutely required P thetase domain and the change in cofactor specificity from NADP to NAD can bind in close proximity to the 2-hydroxyl of NAD through (4). This change in cofactor specificity is important because the use of NAD 166 198 interactions with Arg and Arg . The characterization of rather than NADP in mitochondria shifts the equilibrium of the reaction to 166 190 198 166 mutants of Arg ,Asp , and Arg show that Arg is primarily favor the production of formyl-THF (5). The increased production of responsible for P binding, while Arg plays a secondary role, formyl-THF is required to meet the demand for glycine and purines during assisting in binding and properly orienting the ion in the cofactor embryogenesis (1, 2, 6). However, it is not known how this cofactor speci- 190 166 binding site. Asp helps to properly position Arg . Mutants of ficity change was accomplished. Asp suggest that the magnesium ion interacts with both P and NMDMC is unique in its absolute requirement for magnesium and the aspartate side chain and plays a role in positioning P and NAD. inorganic phosphate ions for NAD-dependent dehydrogenase activity NMDMC uses P and magnesium to adapt an NADP binding site for and magnesium ions for NADP-dependent dehydrogenase activity (3, NAD binding. This adaptation represents a novel variation of the 7). However, neither ion is essential for the cyclohydrolase activity. The classic Rossmann fold. role of these ions in the dehydrogenase activity is not clear. The sequence of binding of the cofactors and substrates to NMDMC, as established kinetically by Yang and Mackenzie (3) and Rios-Orlandi and During embryogenesis and tumorigenesis mammalian mitochondria MacKenzie (7), suggests a role for P and Mg in the binding of the use a folate-dependent pathway to generate both glycine and one-carbon cofactor; the ions bind to the protein first, followed by NAD and then units to support cytoplasmic purine synthesis (1, 2). One of the enzymes in the folate substrate. A preferred order of binding of the ions was not this pathway, the NAD-dependent methylenetetrahydrofolate dehydro- 5 established; either ion appears to be able to bind to the enzyme and genase-methenyltetrahydrofolate cyclohydrolase (NMDMC), catalyzes affect the binding of the other. These results suggested a possible inter- the interconversion of 5,10-methylenetetrahydrofolate (methylene-THF) action between the two ions in the binding site. and 10-formyltetrahydrofolate (formyl-THF) in mammalian mitochon- The observation that P competitively inhibits the cofactor in NADP-de- pendent dehydrogenase assays of NMDMC led to the proposal that P may * This work was supported in part by Canadian Institutes of Health Research (CIHR) Grant occupy a position adjacent to the 2-hydroxyl of NAD, close to the space 29814. The costs of publication of this article were defrayed in part by the payment of that would be occupied by the 2-phosphate of NADP (3). Previous work on page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. the DC domain of the human NADP-dependent DCS identified two resi- The atomic coordinates and structure factors (code 1ZN4) have been deposited in the Protein 173 197 dues (Arg and Ser ) as being important to the binding of NADP to the Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). enzyme through its 2-phosphate (8). Sequence alignments of mitochon- Supported by the CIHR, Fonds de Recherche en Sante´ du Quebe´c, and McGill 166 drial NAD-DCs with trifunctional NADP-DCSs suggest that Arg and University. Arg (numbered from the amino-terminal glutamate of the mature Supported by the International Center for Diffraction Data. Recipient of a Canada Research Chair in Structural Biology. enzyme) may interact with P (9 and Fig. 1). 4 i To whom correspondence should be addressed: Dept. of Biochemistry, McGill Univer- The crystal structure of the DC domain of the human NADP-dependent sity, McIntyre Medical Sciences Bldg., 3655 Promenade Sir William Osler, Montre´al, Que´bec H3G 1Y6, Canada. Tel.: 514-398-7270; Fax: 514-398-7384; E-mail: DCS has been determined both with bound NADP and with bound NADP [email protected]. and folate analogues (10, 11). The structure of the Escherichia coli NADP- The abbreviations used are: NMDMC, NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase; methylene-THF, 5,10- dependent DC has been determined by x-ray crystallography in the absence methylenetetrahydrofolate; formyl-THF, 10-formyltetrahydrofolate; DCS, methyl- of bound substrates (12), and the structure of the Saccharomyces cerevisiae enetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase- formyltetrahydrofolate synthetase; DC, methylenetetrahydrofolate dehydrogenase- NAD-dependent dehydrogenase has been determined with and without methenyltetrahydrofolate cyclohydrolase; GR, glutathione reductase; DH, bound NAD (13). However, since no crystal structure of NMDMC has yet dehydrogenase; PDB, Protein Data Bank; MOPS, 4-morpholinepropanesulfonic acid; WT, wild type. been obtained, we constructed a homology model of the enzyme based on 34316 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 This is an Open Access article under the CC BY license. Magnesium and Phosphate Binding Sites of NMDMC FIGURE 1. Structural alignment of human NMDMC with the templates used to construct the homology model. The templates are the DC domain of the human NADP-depend- ent DCS crystallized with NADP and a folate analogue (PDB ID: 1DIB), the E. coli NADP-dependent DC (PDB ID: 1B0A), and the S. cerevisiae NAD-dependent dehydrogenase crystallized with NAD (PDB ID: 1EE9). A, alignment of the primary structures of NMDMC and the templates adjusted to reflect the fit of the crystal structures 1B0A and 1EE9 to 1DIB. Residues that are aligned in the sequence are considered structurally homologous.-Helices are marked in red and-sheets in blue. Residues targeted in mutagenesis experiments are marked with an asterisk. B and C, overlay of the NMDMC model and the template structures, showing one monomer of the dimer from two sides. The 1B0A structure is cyan, the 1DIB structure is magenta, the 1EE9 structure is blue, and the NMDMC model is green. three related structures and used this to locate the P and Mg binding sites Construction of the Homology Model of Human NMDMC—Crystal using site-directed mutagenesis. structures for three NMDMC homologues have been determined. The DC domain of the human NADP-dependent DCS structure has been EXPERIMENTAL PROCEDURES solved with NADP (10) and with NADP and three different folate ana- Materials—(R,S)-tetrahydrofolate was synthesized according to the logues (11). The E. coli NADP-dependent DC structure has been solved method of Drury et al. (14) and stored in sealed glass vials at 4 °C. in the absence of ligands (12). The S. cerevisiae NAD-dependent dehy- Nickel-nitrilotriacetic acid-agarose was obtained from Qiagen. Vent drogenase has been solved both without ligands and in complex with DNA polymerase was obtained from New England Biolabs, and restric- NAD (13). Templates with bound cofactors and substrates were tion enzymes were products of New England Biolabs and MBI Fermen- selected to build a holoenzyme model of human NMDMC. The three tas. Oligonucleotide primers were obtained from Sigma Genosys, Qia- template structures were obtained from the Protein Data Bank (PDB): gen Operon, and Integrated DNA Technologies, Inc. NAD, imidazole, the DC domain of the human NADP-dependent DCS in complex with and rifampicin were purchased from Sigma. (R,S)-methenyltetrahydro- NADP and a folate analogue (PDB ID: 1DIB), the E. coli NADP-depend- folate was obtained from B. Schircks Laboratories (Jona, Switzerland). ent DC (PDB ID: 1B0A), and the S. cerevisiae NAD-dependent dehydro- All other chemicals and reagents were purchased from Bioshop or BDH genase in complex with NAD (PDB ID: 1EE9). Although the sequence and were of analytical grade. similarity among these proteins is not particularly high as the pair-wise OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34317 Magnesium and Phosphate Binding Sites of NMDMC TABLE ONE Dehydrogenase activity of Arg mutants Specific activity (% WT) Enzyme NAD-DH NADP-DH 1 1 mol min mg WT 22.5  1.1 (100) 2.92  0.06 (100) R166A ND ND R166S ND ND R166K ND ND a 1 1 ND  not detectable (0.01 mol min mg ). TABLE TWO Dehydrogenase activity of Asp mutants Specific activity (% WT) Enzyme NAD-DH NADP-DH 1 1 mol min mg WT 22.5  1.1 (100) 2.92  0.06 (100) D190A ND ND D190S ND ND D190E 0.024  0.001(0.1) ND FIGURE 2. Ribbon diagram of NMDMC homology model with docked NAD and inor- ganic phosphate. D190N 3.64  0.03 (16) 3.96  0.07 (136) a 1 1 ND  not detectable (0.01 mol min mg ). optimization residues 243 and 253 were fixed to keep their position homologous to the 1DIB structure. NAD, P , and NADP were docked into the model based on the position of the cofactors in the template structures. The crystal structures 1B0A and 1EE9 show only one mon- omer of the enzymes and so the resulting model showed only one mon- omer of NMDMC, which is known to be a dimer (21). To model the dimer two copies of the model were superimposed on the 1DIB struc- ture using Lsqman (16), and the geometry and stereochemistry of the side chains along the dimer interface was optimized using CNS (19). The quality of the model was evaluated at every step using Procheck (22). Pictures of the model were generated using Pymol (23). FIGURE 3. Comparison of the cofactor binding sites of NMDMC and the DC domain Addition of Six-histidine Tags—To simplify purification a COOH- of the human DCS. A, the NAD and inorganic phosphate binding site of the NMDMC 166 198 133 190 model. Arg and Arg are found in monomer A and Asp and Asp in monomer B. terminal six-histidine tag was added to NMDMC by PCR using primers B, the NADP binding site of the 1DIB structure. including the 3 terminus of the required coding region of the cDNA, the sequence of the tag and an XhoI site. The histidine-tagged cDNA percent identity ranges between 22–43%, the three-dimensional struc- was then subcloned into pBKeHB1 (24) to make pBKeHB1 303H6. The tures are highly homologous (see Fig. 1). six-histidine tag replaces an unstructured tail made up of twelve resi- A multiple sequence alignment of the target and template sequences dues that have no influence on enzyme activity. The kinetic constants was generated using ClustalW (15). The alignment was then refined to and activities of 303H6 are identical to the full-length, non-histidine- include structural data using alignments to the 1DIB structure gener- tagged NMDMC (data not shown), and so the histidine tag does not ated using a brute force structural alignment method followed by iter- interfere with the enzyme function. The truncated version of the pro- ative cycles of improvement as implemented in the program Lsqman tein was selected for this study because it had better expression levels (16). Structural elements for the model were selected from the tem- than full-length NMDMC with six histidines added and to circumvent plates by highest sequence identity to NMDMC. The structural ele- any problems that may arise from having the six-histidine tag added to ments from the aligned templates were linked together and the side the mobile unstructured tail. For simplicity, in the results section 303H6 chains mutated using the computer program O (17) with the Lovell et al. is referred to as wild type, meaning unmutated pBKeHB1 303H6. (18) rotamer data base. Site-directed Mutagenesis—Mutations were introduced into The loop from residue 182 to 190, unique to mitochondrial NAD-de- pBKeHB1 303H6 using in vitro overlap extension PCR as in Sundarara- pendent DCs (9 and Fig. 1), lacks structural data and was not included in jan and MacKenzie (20). The entire insert of the resulting vector was the final structure. The position of Asp suggested that it could play a sequenced by automated sequencing (Genome Que´bec) to confirm the role in the cofactor binding site, so it was included in the model, and its integrity of each mutant. position was determined by local energy minimization using CNS (19). All of the mutants in this study should affect the NAD(P) cofactor The loop from residue 244 to 252 is disordered in the 1DIB structure binding and thus only the dehydrogenase activity, leaving the cyclohy- and is not present in the E. coli and S. cerevisiae enzymes. This loop has drolase activity and folate substrate binding kinetic constants as con- been shown not to be required for DH activity or cofactor binding (20) trols for gross disruptions of the protein structure. All of the mutants and so was not included in the model. The geometry and stereochem- reported in this study retained significant cyclohydrolase activity. There istry of the model structure was optimized using CNS (19). For the were no significant differences between the K values for methylene- 34318 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 Magnesium and Phosphate Binding Sites of NMDMC TABLE THREE Kinetic constants of D190N compared to wild-type Phosphate Mg (NAD) K Enzyme K of NADP-DH K K n NAD Mg (NADP) NADP i m mM M M M WT 4.13 412  55 351  21 1.99  0.10 202  13 321  36 352  41 D190N 3.43 3100  570 2050  130 1.62  0.11 3880  620 1990  460 689  121 THF, with either NAD or NADP as the cofactor, or for methenyl-THF TABLE FOUR for any of the mutants. This shows that there are no gross disruptions of Dehydrogenase activity of Arg mutants the protein structure in the mutants and that the folate substrate bind- Specific activity (% WT) ing site is unaffected by these mutations. Enzyme NAD-DH NADP-DH Protein Expression and Purification—pBKeHB1 303H6 constructs 1 1 mol min mg expressing histidine-tagged NMDMC were transformed into E. coli WT 22.5  1.1 (100) 2.92  0.06 (100) BL21 DE3. Overnight cultures of transformed bacteria were used to R198A 0.170  0.032 (0.8) 0.156  0.021 (5.3) inoculate 100 ml of Terrific Broth supplemented with 200 g/ml ampi- R198S 0.207  0.018 (0.9) 1.54  0.09 (53) cillin and incubated at 37 °C with shaking at 250 rpm. Expression was R198K 12.6  1.2 (56) 1.24  0.05 (42) induced when cultures reached an A 1.0 to 1.2 by adding isopropyl 1-thio--D-galactopyranoside to a final concentration of 2 mM. After 30 Schmidt et al. (11), typically using four time points at 2-min intervals min 15 g/ml rifampicin was added to inhibit host translation. Cells and at least five variable substrate concentrations. were harvested after an additional 90 min by centrifugation for 20 min at Cyclohydrolase assays were performed as described in Pawelek and 4000 rpm at 4 °C in a Sorvall RC-3. Pellets of 0.3 to 0.4 g cells were MacKenzie (26), with 5 mM magnesium chloride added to the buffer. stored at 85 °C. Results reported are the average of three separate determinations done Frozen pellets were thawed on ice and resuspended in 10 ml of son- in triplicate. The determination of kinetic constants was done as for the ication buffer (0.1 M potassium phosphate (pH 7.3), 35 mM -mercap- standard cyclohydrolase assay, using at least five methenyl-THF toethanol, 1 mM benzamidine hydrochloride, and 1 mM phenylmethyl- concentrations. sulfonyl fluoride). Resuspended cells were disrupted by sonication on For enzymes with sufficient activity, initial rate data were fitted to the ice using 10–12 pulses of 10 s each separated by intervals of 1 min. Michealis-Menten equation by non-linear regression using Sigmaplot Lysates were cleared by centrifugation at 12,500 rpm at 4 °C in a Sorvall (Systat Software Inc.). The kinetic constants for Mg with NAD as a SS-34 rotor for 20 min. Protamine sulfate (0.1 volume of 10 mg/ml cofactor were determined in the same manner by fitting to the Hill solution) was added to the supernatant, followed by an additional equation. Standard errors of the fit for K and V were under 25%. 20-min centrifugation at 12,500 rpm. m max Results are reported as the average and standard deviation of three to The protamine sulfate-treated crude extract was adjusted to contain five separate K determinations. 0.5 M NaCl and 15 mM imidazole and added to 10 ml of a 50% slurry of m Inhibition of NADP-dehydrogenase activity by P was examined by Ni-NTA-agarose resin in binding buffer (0.1 M potassium phosphate performing assays at four fixed P concentrations at varying concentra- (pH 7.8), 0.5 M NaCl, 15 mM imidazole, 1 mM phenylmethylsulfonyl i tions of NADP, similar to Yang and MacKenzie (3). K values for P fluoride, 1 mM benzamidine, 10 mM -mercaptoethanol, and 20% (v/v) i i against NADP were calculated from the intercepts of replots of the glycerol). The slurry was mixed for 1.5 h at 4 °C on a rotator, and the slopes of Lineweaver-Burk plots versus P concentration. resin was collected by centrifuging at 1,000 rpm for 10 min in a Sorvall RC-3. The resin was then resuspended in 5 ml of binding buffer and RESULTS packed into a 1.5-cm diameter column. The column was washed once at NMDMC Homology Model 0.25 ml/min with 3 volumes of binding buffer, followed by 3 volumes of binding buffer containing 50 mM imidazole. The enzyme was eluted at The homology model of NMDMC is in good agreement with the 0.1 ml/min with binding buffer containing 250 mM imidazole. Fractions template structures. The root mean square deviation of the  carbons containing the enzyme were identified by Bradford assay (25). All between the model and each template is as follows: 1DIB structure enzyme preparations were evaluated for purity by SDS-PAGE on 10% (human DCS), 1.23 Å; 1B0A structure (E. coli DC), 0.92 Å; and 1EE9 polyacrylamide gels. Protein concentration was determined by Bradford structure (S. cerevisiae D), 1.40 Å (see Fig. 1). The Ramachandran plot of assay performed in triplicate, using bovine serum albumin as a standard. the model demonstrates that 82.6% of residues are in core regions, 14.6% Enzyme Assays and Kinetics—Dehydrogenase assays were performed in allowed regions, and 2.8% in generously allowed regions. No residues after Yang and Mackenzie (3) and Pawelek and MacKenzie (26). Stand- are found in disallowed conformations. A ribbon diagram of the homol- ard conditions buffer contained 25 mM MOPS (pH 7.3), 5 mM potassium ogy model, shown with docked P and NAD, is shown in Fig. 2. The phosphate (pH 7.3), 5 mM magnesium chloride, 2.5 mM formaldehyde, coordinates for the model have been deposited in the Protein Data Bank 0.2 mM (6R,S)-tetrahydrofolate, 36 mM -mercaptoethanol, and 0.6 mM with accession number 1ZN4. NAD. For NADP-dependent DH assays the assay mixture was modified The Phosphate Binding Site of NMDMC by removing the potassium phosphate and NAD and including 2 mM NADP. Standard activity assays are reported as the average of three The competitive inhibition of P against NADP in dehydrogenase separate determinations performed in triplicate using a single fixed time assays lead Yang and MacKenzie (3) to propose that P binds to point. The determination of kinetic constants was performed on NMDMC in a position analogous to the location of the 2-phosphate of mutants with sufficient activity using a multiple time point assay as in NADP in DCS. The NAD binding site of the NMDMC model is shown OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34319 Magnesium and Phosphate Binding Sites of NMDMC TABLE FIVE Kinetic constants of Arg mutants compared to wild-type Phosphate Mg (NAD) K Enzyme K of NADP-DH K K n NAD Mg (NADP) NADP i m mM M M M WT 4.13 412  55 351  21 1.99  0.10 202  13 321  36 352  41 R198S 10.2 ND ND ND ND 349  49 255  47 R198K 9.35 2230  530 1470  100 1.49  0.08 918  190 1110  80 2120  20 ND  not determined due to insufficient enzyme activity. 190 166 residue that disrupted the ionic interaction of Asp and Arg (such TABLE SIX as D190A and D190S) or resulted in a shift of the position of Arg in Dehydrogenase activity of Asp mutants the binding site (D190E) resulted in the loss of DH activity. Only D190N Specific activity (% WT) retained significant amounts of DH activity with both cofactors. This Enzyme NAD-DH NADP-DH mutant would have a weaker interaction between residues 166 and 190, 1 1 mol min mg 166 166 allowing greater movement of Arg , but the position of Arg would WT 22.5  1.1 (100) 2.92  0.06 (100) not shift due to the similar size of the Asp and Asn side chains. The D133A 0.392  0.037 (2) 0.160  0.028 (6) affinity of D190N for P , as measured by its inhibition constant against D133S 3.99  0.33 (18) 1.01  0.06 (35) NADP, is not changed although all values of K for substrates and ions D133E ND ND are elevated. These changes in the K values most likely reflect the D133N 4.59  0.24 (20) 2.40  0.15 (82) inhibition of DH activity due to the difficulty of positioning the ligands a 1 1 ND  not detectable (0.01 mol min mg ). properly in the binding site when Arg , and therefore P , are free to 2 2 move. The increase in the Mg constants suggests that the Mg and P compared with the NADP binding site of DCS in Fig. 3. The P modeled binding sites, like the NAD and P binding sites, interact with each other. in the NMDMC structure is slightly displaced from NAD into a posi- The K for NADP is less affected than that for NAD, because the 166 198 tively charged cavity made up of Arg and Arg . Three of the P i 2-phosphate is covalently attached and requires positioning of a single oxygens are now within hydrogen bonding distance (2.5–3.5 Å) of the ligand rather than two separate molecules. Binding NADP might help to 166 198 2- and 3-hydroxyls of the NAD ribose. Arg and Arg are homol- partially reposition Arg in the D190N mutant. These mutations show 173 197 190 166 ogous to Arg and Ser , respectively, of the human DCS, which are that the role of Asp is to position Arg in the binding site. 198 198 involved in NADP binding (8). Mutagenesis of Arg —The properties of Arg mutants are shown Arg of DCS forms multiple hydrogen bonds with and stabilizes the in TABLES FOUR and FIVE. In the case of R198A, where the arginine is charge of the 2-phosphate of NADP (10). When this residue is mutated, substituted with a residue that is not capable of forming hydrogen dehydrogenase activity of DCS is reduced to less than 2.5% of wild-type bonds, both NAD-DH and NADP-DH activities are significantly activity, and values of K for NADP are greatly increased, showing the reduced, indicating an important role for this residue (TABLE FOUR). importance of this residue in binding the cofactor (8). In the homology When this arginine is substituted by serine, which is the homologous model (Fig. 3) Arg appears to have the potential to form multiple residue in DCS, NAD-DH activity is drastically reduced, while the hydrogen bonding interactions with P and can also contribute to stabi- NADP-DH is relatively unaffected. The P affinity, as measured by the K 173 i i lizing the charge of P , similar to the role of Arg in DCS. Therefore this for P , is reduced in this mutant, which shows that Arg does assist in residue was targeted for mutagenesis. P binding. The K values for Mg and NADP are not affected by this 197 i m Ser of the human DCS forms one hydrogen bond with the 2-phos- mutation, which shows that the binding site of Mg is not distorted in phate of NADP (10) and was shown by mutagenesis to play a supporting 166 2 R198S and that the combination of Arg and Mg is sufficient to role in cofactor binding (8). In NMDMC (Fig. 3) Arg is the homolo- allow wild-type affinity for NADP. gous residue to Ser of DCS. In the homology model of NMDMC it The R198K mutant, in which the arginine is replaced with a smaller appears that this residue has the potential to form hydrogen bonds with and more flexible residue that also has a positive charge, retains roughly P and can also contribute to charge stabilization and so it too was 50% of both NAD and NADP-dependent DH activities. In R198K all of targeted for mutagenesis. 190 the K values are significantly increased compared with wild type. The Upon examination of the model, the side chain of Asp , a residue 166 K values in this case likely reflect both the loss of affinity for P (shown m i unique to NMDMC, was observed behind Arg in a position that by the larger K value for P ), and the difficulty of properly positioning i i suggests an electrostatic interaction (Fig. 3). Mutation of this residue the P in the binding site due to the increased flexibility of the side chain. provides the opportunity to manipulate the position of Arg in the The Mg constants, as in D190N, are also affected by this mutation, protein and observe the effects, without actually changing the nature of again showing that the two binding sites interact with each other. In this the side chain. 166 166 case, in contrast to R198S, the bulky side chain may be occluding the Mutagenesis of Arg —Even conservative mutations of Arg abro- Mg site or distorting it in a way that impairs binding. gate dehydrogenase activity (TABLE ONE). This is consistent with the 173 173 The role of Arg is illustrated by the effects of mutations on the K role of Arg in DCS. Arg is primarily responsible for NADP binding value for P . Although R198S and R198K have roughly the same affinity through the 2-phosphate, which suggests that Arg has a similar role i for Pi, as measured by the K , R198S almost totally lacks NAD-DH activ- in binding P in NMDMC. Mutagenesis of Asp —To examine the effects of changing the posi- ity, whereas R198K retains greater than 50%. Simply binding the phos- 166 190 tion of Arg without altering the side chain Asp was mutated. The phate is insufficient suggesting it must also be positioned properly for assay results are shown in TABLES TWO and THREE. Mutations of this DH activity by an electrostatic interaction with a positive charge. Arg 34320 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 Magnesium and Phosphate Binding Sites of NMDMC TABLE SEVEN Kinetic constants of Asp mutants compared to wild-type Phosphate Mg (NAD) K Enzyme K of NADP-DH K K n NAD Mg (NADP) NADP i m mM M M M WT 4.13 412  55 351  21 1.99  0.10 202  13 321  36 352  41 D133S 2.60 1320  180 2780  420 1.83  0.55 2170  190 3440  570 503  43 D133N 2.95 1250  240 2450  80 1.22  0.00 1990  300 3680  570 580  40 positions the phosphate that interacts with Arg and orients it within the binding site to interact with NAD. The Magnesium Binding Site Kinetics with Magnesium—A more sensitive multiple time point assay was used to determine K values for this study, as opposed to the single time point assay used previously (3). This method revealed a previously unobserved co-operativity in the binding of Mg with NAD. The velocity versus [Mg ] curve with NAD as a cofactor is sigmoidal and fits to the Hill equation. The value of n for 303H6 was found to be 1.99 0.10 indicating near perfect co-operativity of binding. This effect is not observed in velocity versus [Mg ] curves using NADP as a cofac- tor nor when [P ] is the variable substrate. Consequently both K and n FIGURE 4. The magnesium ion binding site may be located in a cavity bounded by values are reported for magnesium. The significance of this apparent Asp , the inorganic phosphate, and the NAD cofactor, which could provide co- co-operativity is not clear. ordination points for the cation. Identification of Potential Magnesium Binding Sites—Potential Mg 166 198 binding sites were identified by examining a multiple sequence align- the human DCS. In NMDMC, Arg and Arg provide a positively ment of methylene-THF dehydrogenases to find aspartate and gluta- charged pocket for binding P . Arg is the residue that is primarily mate residues that are conserved in human, mouse, and fruit fly responsible for binding P ; all mutants of this residue, even the conserv- NMDMC but not conserved in NADP-dependent enzymes that do not ative mutation to lysine, lack dehydrogenase activity. The lack of activity require Mg . These residues in particular were selected because they of R166K is not that surprising given that arginine residues are often are the preferred interacting partners of Mg , at least one of them favored over lysine at P sites because they can form multiple interac- being found in all known Mg binding sites (27). This search narrowed tions with the ion and can be resonance stabilized (28, 29). down the potential interacting residues to 8 from 36. The local environ- Mutagenesis of Asp shows that this residue is required to properly ment of these residues was examined in the homology model to deter- position Arg in the binding site. This type of arginine-aspartate inter- mine whether any could contribute to a Mg binding site. The exist- action to position the side chain has been previously observed in other ence of a possible binding partner to permit charge balance, the size of proteins that bind phosphate ions (30, 31, 32). Mutation of Asp to the the potential site (5–6 Å distance between binding partners), the larger glutamate residue displaces Arg and essentially inactivates charge of the site, and its access to the solvent were evaluated. Of the NMDMC. The D190N mutation retains significant DH activity. How- eight residues, only four are situated such that they could be part of a ever, the weakened interaction with Arg allows the residue to move, Mg binding site. Preliminary mutagenesis experiments eliminated impeding ligand binding, which increases the ligand K constants. 133 166 three of these residues leaving Asp as the only residue likely to con- These mutations show that the position of Arg is critical for cofactor tribute to a Mg binding site. binding to NMDMC. 133 133 198 166 198 Mutagenesis of Asp —The properties of the Asp mutants are Arg Positions Phosphate in the Binding Site—Unlike Arg , Arg shown in TABLES SIX and SEVEN. Substitution of aspartate by gluta- can be mutated to other residues without losing all dehydrogenase mate completely inactivates the DH activity with both NAD and NADP activity. The dehydrogenase activities and kinetic constants of Arg cofactors, indicating that the position of the carboxyl group is critical. mutants with NAD and NADP show that this residue assists in P bind- However, D133A, D133S, and D133N, without free carboxyl groups, ing but is not essential for the binding of NADP. A comparison of the K retain some DH activity. The D133S and D133N mutants show no for P and dehydrogenase activities of R198S and R198K establishes that reduction in affinity for P , as indicated by the K values, but the values of the role of Arg is not only to assist in binding P to NMDMC but also i i i 2 133 K for Mg are greatly elevated. These results support a role for Asp to position P within the binding site to optimize the interactions that m i 2 2 198 190 in helping to bind Mg . The loss of Mg affinity in these mutants allow NAD to bind. The Arg and Asp mutants both suggest that affects the positioning of P in the binding site and results in elevated the position of the phosphate is critical when it is not covalently bound 166 198 kinetic constants for all the ligands. This suggests that the role of the to the dinucleotide cofactor. The mutagenesis of Arg and Arg , Mg ion is to assist in the binding and positioning of P , much like the which are homologous to the 2-phosphate binding residues of DCS, role of Arg . confirms the hypothesis of Yang and MacKenzie (3) that P binds to NMDMC near the 2-hydroxyl of NAD. P in this position can form DISCUSSION multiple hydrogen bonds with the NAD cofactor. The K values for The Position of Arg Is Critical for Phosphate Binding—The model NAD and NADP are similar in the wild-type enzyme, but the V for max 166 198 of NMDMC shows that residues Arg and Arg are homologous to the NADP activity is only 20% of that with NAD and P .WhenNADis 173 197 Arg and Ser that interact with the 2-phosphate of NADP bound to bound to NMDMC the hydrogen bond length between P , and the OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34321 Magnesium and Phosphate Binding Sites of NMDMC FIGURE 5. Comparison of the cofactor binding sites of alcohol dehydrogenase (PDB ID: 1HDX) (A), NMDMC (B), and the DC domain of the human DCS (C) (PDB ID: 1DIB). The diphosphate binding domain of NMDMC lacks the close contact of protein and cofactor that is seen in the classic NAD binding site of alcohol dehydrogenase. This domain is more similar to the DC domain of DCS, which is entirely dependent on interactions with the 2-phosphate of NADP for cofactor binding (8). The magnesium and inorganic phosphate ions in the cofactor binding site of NMDMC set up a web of ionic and hydrogen bonding interactions that compensate for the lack of the covalent bond with the phosphate. This allows the adaptation of an NADP binding site to bind NAD. ribose moiety is longer than the bond length of the 2-phosphate of been mutated by Scrutton et al. (36) to preferentially use NADH. In that NADP. Therefore, when bound to the P site of NMDMC, NADP will protein seven residues, including the arginines, were mutated to alter shift upwards in the binding site, moving the nicotinamide moiety the cofactor binding region to more strongly resemble a classic NADH slightly out of position, affecting the activity of the enzyme but not the binding site. affinity for NADP. The interaction of Mg and P with a ligand containing a sugar The Magnesium and Phosphate Ions Interact—Mutagenesis of the moiety is similar to the crystal structure of the rat 3-phosphoadenosine residues of the P binding site supports the interaction of the P and 5-phosphate/inositol 1,4-bisphosphate phosphatase with bound AMP, i i 2 2 2 Mg binding sites suggested earlier by the enzyme kinetics (3). Muta- P , and Mg ions (37). In this structure three Mg ions bind the P , i i tions such as D190N and R198K that alter the positioning of the P in the which forms hydrogen bonds with the 2- and 3-hydroxyls of the ribose binding site also result in reduced affinity for Mg and indicate an moiety. interaction of the binding sites. The R198S mutant, which has decreased The Structure of the Cofactor Binding Site Is Similar to That of NADP P affinity (elevated K ) without altering NADP binding, has no effect on Sites—When the cofactor binding site of NMDMC is compared with a i i Mg affinity. These results suggest that the phosphate ion itself might classic Rossmann NAD binding site and to the NADP binding site of make up part of the Mg binding site and suggests that the role of the DCS, the role for the ions becomes more apparent (Fig. 5). The classic Mg ion is to assist in the binding and positioning of P , much like the NAD site has multiple interactions between the cofactor and the protein role of Arg . to enhance cofactor binding. The GXGXXG consensus sequence inter- 2 133 AMg ion interacting with Asp could co-ordinate with P within acts with the pyrophosphate moiety and maintains close proximity to the cavity illustrated in Fig. 4. The Mg binding site of NMDMC is the cofactor to maximize interactions. In particular, the second glycine made up of Asp and P , and these negative charges provide the charge in this region is thought to be important for close contact because any balance for the ion. The remaining four co-ordination points of the side chain at this position would disrupt cofactor binding (35). NAD- Mg (27) are most likely provided by backbone carbonyl groups, the binding proteins also typically have a conserved aspartate residue that 3-hydroxyl of NAD, and water. These interactions place the Mg forms hydrogen bonds with the hydroxyl groups of the adenine ribose of within a box with corners at the carboxyl group of Asp , the P , the NAD (35, 38). In contrast, NADP binding sites depend on the interac- 166 2 carbonyl of Arg , and the 3-hydroxyl of NAD. The position of Mg tion of the 2-phosphate of NADP with an arginine side chain (38), as is suggests that the ion stabilizes the position of P and NAD in the binding the case with DCS (8, 10). The GXGXXG consensus sequence is not as site through hydrogen bonds and charge interactions. The P stabiliza- strictly conserved in NADP-binding proteins, and the aspartate residue tion role in NMDMC is similar to the role of Mg in many proteins that is no longer conserved. NMDMC and DCS share the consensus use ATP or other phosphorylated substrates or intermediates (33). sequence of GRSXXXG (residues 172–178 of DCS and 165–171 of Similarities to P and Mg Usage in GR and Rat 3-Phosphoad- NMDMC). The substitution of the second glycine by serine in this enosine 5-Phosphate/Inositol 1,4-Bisphosphate Phosphatase—The use region disrupts the close interactions usually required for NAD binding of P to help bind NAD in NMDMC is similar to NADH binding to (Fig. 5). NMDMC also lacks the conserved aspartate residue. Thus, the native glutathione reductase (GR; Ref. 34). GR preferentially uses cofactor binding site of NMDMC more closely resembles an NADP NADPH to reduce oxidized glutathione to glutathione. NADPH binds binding site than a classic NAD binding site. Mutagenesis experiments to GR through interactions with two arginines (218 and 224). GR can on DCS showed that NADP binding to the protein is almost entirely also use NADH as a cofactor; however, the affinity of GR for NADH is dependent on the interaction between the 2-phosphate and Arg (8); roughly 60-fold weaker than the affinity for NADPH, and it can only the other small interactions between the protein and the cofactor were bind to the protein in the presence of P (34). The P in GR binds roughly not sufficient for NADP binding. Given the similarity of the NMDMC i i in the same position as the 2-phosphate of NADPH, interacting with and DCS cofactor binding sites, it seems clear that the role of the ions in the two arginine side chains. Although the use of the two arginines is NMDMC is to compensate for the lack of a covalently bound phosphate common between GR and NMDMC, these motifs are not structurally group on the cofactor. The Mg and P ions mediate multiple hydrogen related; it is not possible to overlay these motifs in the structures. In GR bonding interactions that adapt an NADP site to bind NAD. the arginines are independent of the GXGXXG motif (residues 174– Several laboratories have attempted to engineer NADP-specific pro- 179) that interacts with the diphosphate moiety of NAD(P)H (35), teins to preferentially use NAD (36, 39, 40). These groups have used a whereas in NMDMC Arg is the second residue of the motif. GR has mutagenesis approach to alter multiple side chains around the cofactor 34322 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 40 •OCTOBER 7, 2005 Magnesium and Phosphate Binding Sites of NMDMC 16. Kleywegt, G. J. (1996) Acta Crystallogr. Sect. D Biol. Crystallogr. 52, 842–857 binding site to mimic the binding site of a homologous protein specific 17. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. for NAD. Nature has, through evolution, used an entirely different A 47, 110–119 approach to change the cofactor specificity of the mitochondrial meth- 18. Lovell, S. C., Word, J. M., Richardson, J. S., and Richardson, D. C. (2000) Proteins 40, ylene-THF dehydrogenase, producing a protein whose specificity for 389–408 19. Brunger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, NAD compares favorably to these engineered proteins (36, 39, 40). This R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., use of Mg and P to bind NAD to the active site of NMDMC repre- Simonson, T., Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, sents a novel variation of the Rossmann fold. 905–921 20. Sundararajan, S., and MacKenzie, R. E. (2002) J. Biol. Chem. 277, 18703–18709 Acknowledgments—We thank Peter Pawelek and Saravanan Sudararajan for 21. Mejia, N. R., Rios-Orlandi, E. M., and MacKenzie, R. E. (1986) J. Biol. Chem. 261, 9509–9513 helpful discussion. 22. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. 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(1994) Nucleic Acids Res. 22, 4673–4680 278, 49203–49214 OCTOBER 7, 2005• VOLUME 280 • NUMBER 40 JOURNAL OF BIOLOGICAL CHEMISTRY 34323

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Oct 7, 2005

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