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

Learn More →

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues *

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 22, pp. 15507–15517, May 30, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues Received for publication, February 3, 2014, and in revised form, April 14, 2014 Published, JBC Papers in Press, April 14, 2013, DOI 10.1074/jbc.M114.555573 Minhye Shin, Joshua D. Bryant, Jessica Momb, and Dean R. Appling From the Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712 Background: Mitochondria produce one-carbon units for cytoplasmic nucleotide and methyl group synthesis. Results: MTHFD2L uses both NAD and NADP and is expressed in embryonic tissues during neural tube closure. Conclusion: This cofactor specificity allows for rapid response to changing metabolic conditions. Significance: These findings help explain why mammals possess two distinct mitochondrial isozymes that switch expression during neural tube closure. Mammalian mitochondria are able to produce formate from cytoplasm, where the formate is reattached to tetrahydrofolate one-carbon donors such as serine, glycine, and sarcosine. This (THF) for use in de novo purine biosynthesis or further reduced pathway relies on the mitochondrial pool of tetrahydrofolate for either thymidylate synthesis or remethylation of homocys- (THF) and several folate-interconverting enzymes in the mito- teine to methionine. The 1C unit interconverting activities rep- chondrial matrix. We recently identified MTHFD2L as the resented in Fig. 1 by reactions 1–3 (1m–3m in mitochondria) enzyme that catalyzes the oxidation of 5,10-methylenetetrahy- are catalyzed by members of the methylenetetrahydrofolate drofolate (CH -THF) in adult mammalian mitochondria. We dehydrogenase (MTHFD) family in eukaryotes. The cytoplas- show here that the MTHFD2L enzyme is bifunctional, possess- mic MTHFD1 protein is a trifunctional enzyme possessing ing both CH -THF dehydrogenase and 5,10-methenyl-THF 10-formyl-THF (10-CHO-THF) synthetase, 5,10-methenyl- cyclohydrolase activities. The dehydrogenase activity can use THF (CH -THF) cyclohydrolase, and 5,10-methylene-THF either NAD or NADP but requires both phosphate and Mg (CH -THF) dehydrogenase activities (Fig. 1, reactions 1–3). when using NAD . The NADP -dependent dehydrogenase In contrast to the single trifunctional enzyme found in the activity is inhibited by inorganic phosphate. MTHFD2L uses the cytoplasm, three distinct MTHFD isozymes, encoded by three mono- and polyglutamylated forms of CH -THF with similar distinct genes, are now known to catalyze reactions 1m–3m catalytic efficiencies. Expression of the MTHFD2L transcript is (Fig. 1) in mammalian mitochondria. The final step in the mam- low in early mouse embryos but begins to increase at embryonic malian mitochondrial pathway to formate (Fig. 1, reaction 1m) day 10.5 and remains elevated through birth. In adults, is catalyzed by MTHFD1L, a monofunctional 10-CHO-THF MTHFD2L is expressed in all tissues examined, with the highest synthetase (2). The CH -THF dehydrogenase reaction (Fig. 1, levels observed in brain and lung. reaction 3m) is catalyzed by two homologous enzymes, MTHFD2 and MTHFD2L. MTHFD2 was initially identified in 1985 as an NAD -dependent 5,10-methylene-THF dehydro- Folate-dependent one-carbon (1C) metabolism is highly genase (3). Upon purification, this protein was found to be a bifunctional enzyme (4), also possessing CH -THF cyclohy- compartmentalized in eukaryotes, and mitochondria play a critical role in cellular 1C metabolism (reviewed in Ref. 1). The drolase activity (Fig. 1, reaction 2m). This enzyme, now referred cytoplasmic and mitochondrial compartments are metaboli- to as MTHFD2, has been extensively characterized with respect cally connected by the transport of 1C donors such as serine, to kinetics, substrate specificity, and expression profile (3, 5–10). glycine, and formate across the mitochondrial membranes in a mostly unidirectional flow (clockwise in Fig. 1). In mitochon- In 2011 we reported the discovery of a new mammalian mito- dria, the 1C units are oxidized to formate and released into the chondrial CH -THF dehydrogenase, termed MTHFD2L (11). MTHFD2L is homologous to MTHFD2, sharing 60–65% iden- tity. Recombinant rat MTHFD2L exhibits NADP -dependent * This work was supported, in whole or in part, by National Institutes of Health CH -THF dehydrogenase activity when expressed in yeast (11), Grants F32HD074428 (to J. M.) from the Eunice Kennedy Shriver NICHD and GM086856 (to D. R. A.) from NIGMS. but the enzyme was not purified in that study. In addition, it To whom correspondence should be addressed: Dept. of Molecular Biosci- could not be determined whether MTHFD2L was bifunctional ences, The University of Texas at Austin, Welch Hall A5300, Austin. TX because CH -THF cyclohydrolase assays are unreliable in 78712. Tel.: 512-471-5842; E-mail: [email protected]. The abbreviations used are: 1C, one carbon; THF, tetrahydrofolate; CH - crude extracts. This new isozyme is expressed in adult mito- THF, 5,10-methenyltetrahydrofolate; CH -THF, 5,10-methylenetetrahydro- chondria (11), whereas MTHFD2 is expressed only in trans- folate; 10-CHO-THF, 10-formyltetrahydrofolate; MTHFD, methylenetetra- formed mammalian cells and embryonic or nondifferentiated hydrofolate dehydrogenase; eEF2, eukaryotic translation elongation factor 2; TBP, TATA-box-binding protein. tissues (3). This is an open access article under the CC BY license. MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15507 MTHFD2L Exhibits Dual Redox Cofactor Specificity 5-CHO-THF (MP Biomedicals, Solon, OH) was conducted as described previously (16). Oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, IA). Tetrahydropteroylpentaglutamate (H PteGlu ) was prepared 4 5 by a modified NaBH reduction from the corresponding pteroylpentaglutamate (PteGlu ) (Schircks Laboratories, Jona, Switzerland), as described previously (17). Further preparation of 5,10-CH -H PteGlu was accomplished by incubation with 2 4 5 formaldehyde as described previously (14). Mice—All animals used within this study were maintained according to protocols approved by the Institutional Animal Care and Use Committee of The University of Texas at Austin and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cloning of Rat MTHFD2L—RNA isolation and cDNA con- struction of rat MTHFD2L were conducted as described previ- FIGURE 1. Mammalian one-carbon metabolism. Reactions 1– 4 are in both ously (11). Removal of the mitochondrial targeting sequence the cytoplasmic and the mitochondrial (m) compartments. Reactions 1, 2, from the full-length rat MTHFD2L cDNA was accomplished by and 3, 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase, respectively, are catalyzed by trifunc- PCR amplification using primers rMTHFD2L1–40_forward tional C -THF synthase in the cytoplasm (MTHFD1). In mammalian mitochon- (5-GTCATCACCATCACCATCACGGATCCATGGCGAC- dria, reaction 1m is catalyzed by monofunctional MTHFD1L, and reactions 2m and 3m are catalyzed by bifunctional MTHFD2 or MTHFD2L. Reactions 4 GCGGGCC-3 and rMTHFD2L1–40_reverse (5-ACCTTA- and 4m are catalyzed by serine hydroxymethyltransferase and reaction 5 by GCGGCCGCAGATCTGGTACCCTAGTAGGTGATATTC- the glycine cleavage system. Gray ovals represent putative metabolite trans- T-3) (start codon is in bold, and underlined portions are porters. Hcy, homocysteine; dTMP, thymidylate. complementary to the rat MTHFD2L cDNA). The mitochond- The existence of these two CH -THF dehydrogenases rial targeting sequence (amino acids 1–40) was predicted by MitoProt (18). The resulting PCR-amplified fragment was (MTHFD2 and MTHFD2L) in mammalian mitochondria raises cloned into the pET22b vector (Novagen, EMD Biosciences). several questions. Do the two enzymes differ in their catalytic activity or in their substrate or cofactor specificity? Do they The pET22b-rMTHFD2L1–40 was further subcloned into a differ in their tissue distribution or expression profiles? To YEp24-based yeast expression vector containing the Saccha- romyces cerevisiae MET6 promoter using sequence- and ligat- answer these questions, we report here the purification and ion-independent cloning (17, 19). The YEp24 vector was mod- kinetic characterization of MTHFD2L and its embryonic and adult gene expression profiles. We show that MTHFD2L pos- ified to include a multiple cloning site and an N-terminal His sesses CH -THF cyclohydrolase activity and can use either tag (11). This construct (YEp24-rMTHFD2L1– 40) should produce a protein of 307 residues (N-terminal Met  2 Gly  6 NAD or NADP in its CH -THF dehydrogenase activity. His  298 MTHFD2L residues) with a calculated molecular MTHFD2L is expressed during mouse embryonic develop- ment, and there appears to be a switch from MTHFD2 to mass of 33,180 kDa. MTHFD2L expression at about the time of neural tube closure. Expression and Purification of MTHFD2L—The YEp24- rMTHFD2L1–40 construct was transformed into yeast strain A comparison of the enzymatic characteristics and expression MWY4.4 (ser1 ura3–52 trp1 his4 leu2 ade3–65mtd1) using a profiles of MTHFD2 and MTHFD2L revealed differences that may shed light on the roles of these two isozymes in adult and high efficiency lithium acetate yeast transformation procedure embryonic 1C metabolism. (20, 21). Transformed cells were grown in synthetic minimal medium (YMD) containing 0.7% (w/v) yeast nitrogen base EXPERIMENTAL PROCEDURES without amino acids (Difco Bacto) and 2% (w/v) glucose sup- Chemicals and Reagents—All reagents were of the highest plemented with L-serine (375 mg/liter), L-tryptophan (20 mg/li- commercial grade available. NAD and NADP were pur- ter), L-histidine (20 mg/liter), L-leucine (30 mg/liter), and ade- chased from U. S. Biological (Swampscott, MA) and Sigma, nine (20 mg/liter). Cultures were grown at 30 °C in a rotary respectively. HRP-conjugated goat anti-rabbit IgG was from shaker at 200 rpm and were harvested at 3–4 A by centrifu- Invitrogen, and the ECL Plus chemiluminescence detection kit gation at 8000  g for 5 min at 4 °C. The cell pellet was sus- was from GE Healthcare Life Sciences. Polyclonal antibodies pended in 2 ml of 25 mM Tris-Cl (pH 7.5) containing 1% (w/v) specific for MTHFD2L were produced in rabbits by the Dept. of sodium carbonate, 10 mM -mercaptoethanol, and 1 mM phe- Veterinary Sciences, M. D. Anderson Cancer Center, Bastrop, nylmethanesulfonyl fluoride (PMSF)/gram wet weight. The TX. THF was prepared by the hydrogenation of folic acid suspended cells were disrupted with glass beads using a Fast- (Sigma) using platinum oxide as a catalyst and purification of Prep FP120 cell disrupter (MP Biomedicals) followed by incu- the THF product on a DEAE cellulose column (Sigma) (12, 13). bation for 30 min at 4 °C to facilitate dissociation of MTHFD2L CH -THF was prepared nonenzymatically from THF and from cellular membranes. Cell debris was removed by centrif- formaldehyde (Fisher) (14). The yield of CH -THF was deter- ugation at 30,000  g for 30 min at 4 °C. The extracted mined by solving the equilibria of THF, formaldehyde, and MTHFD2L protein was then dialyzed at 4 °C overnight against -mercaptoethanol (15). The preparation of CH -THF from 25 mM Tris-Cl, 10 mM -mercaptoethanol, 1 mM PMSF, 20% 15508 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity TABLE 1 (v/v) glycerol, and 500 mM KCl at pH 8.5. Concentrated cell Primers used for gene expression profiling of the MTHFD gene family lysate (15 ml) was added to 9 ml of Ni -charged His-Bind in mouse embryos and adult organs resin (Novagen, Darmstadt, Germany) in 15 ml of binding Primer name Sequence Location buffer (final concentration: 25 mM Tris-Cl, 10 mM -mercapto- MTHFD1 F 5-TTCATCCCATGCACACCCAA-3 Exon 6 ethanol, 1 mM PMSF, 20% (v/v) glycerol, 500 mM KCl, and 20 MTHFD1 R 5-ATGCATGGGTGCACCAACTA-3 Exon 7 mM imidazole at pH 8.5). The slurry was mixed for3hat4 °C MTHFD1L F 5-GGACCCACTTTTGGAGTGAA-3 Exon 12 MTHFD1L R 5-ATGTCCCCAGTCAGGTGAAG-3 Exon 14 and then packed into a column. The column was washed at MTHFD2 F 5-ACAGATGGAGCTCACGAACG-3 Exon 5 room temperature with 40 column volumes of wash buffer con- MTHFD2 R 5-TGCCAGCGGCAGATATTACA-3 Exon 6 MTHFD2L F1 5-GGCGGGAAGATCCAAGAACG-3 Exon 6 taining 25 mM Tris-Cl, 10 mM -mercaptoethanol, 1 mM PMSF, MTHFD2L R1 5-CGCTATCGTCACCGTTGCAT-3 Exon 7 500 mM KCl, 10% (v/v) isopropanol, and 60 mM imidazole (pH MTHFD2L F2 5-GGCCAGCAGAGAGAAGAGACT-3 Exon 2 MTHFD2L R2 5-CCATGATTCCACTCCTTGCT-3 Exon 3 8.5) at a flow rate of 2–3 ml/min. His-tagged MTHFD2L was MTHFD2L F3 5-GAGGTGATGCAACGGTGAC-3 Exon 7 eluted at 4 °C at 0.5–1 ml/min with binding buffer containing MTHFD2L R3 5-GAATACCCGCAGCCACTATG-3 Exon 8 eEF2 F 5-CGCATCGTGGAGAACGTCAA-3 Exon 4 250 mM imidazole. Fractions containing active enzyme were eEF2 R 5-GCCAGAACCAAAGCCTACGG-3 Exon 5 determined by assaying for CH -THF dehydrogenase activity 2 TBP F 5-CATGGACCAGAACAACAGCC-3 Exon 2 TBP R 5-TAAGTCCTGTGCCGTAAGGC-3 Exon 3 (see below), and protein concentration was determined using the Bio-Rad protein assay reagent. Purified MTHFD2L was stored at 20 °C in 25 mM Tris-Cl, 10 mM -mercaptoethanol, tions at varying concentrations of NADP . Over the 5-min 100 mM KCl, and 50% (v/v) glycerol at a final pH of 7.5. Purity of period of measurement, 20% or less of the substrate was con- enzyme preparations was evaluated by SDS-PAGE. Enzyme verted to product, ensuring that initial rates were observed. K stability was confirmed by assaying CH -THF dehydrogenase was calculated from a global fit of the data to a competitive activity in aliquots of stored enzyme over a 1-week period. inhibition model using Prism. RNA Isolation and cDNA Synthesis—Tissue was collected The purified enzyme retained 75% 5,10-CH THF dehydro- genase activity after 1 week of storage, and all kinetic analyses from six male and six female C57BL/6 mice between 4 and 6 were performed within 1 week of enzyme purification. weeks of age. Embryos were dissected at the days indicated. For 5,10-Methylene-THF Dehydrogenase and 5,10-Methenyl- embryonic days 8.5–11.5, five embryos were pooled for each THF Cyclohydrolase Assays—A microplate assay was used for time point; for embryonic days 12.5–17.5, three embryos were determination of kinetic parameters as described previously pooled for each time point. Tissue and embryos were washed (22). CH -THF dehydrogenase activity was determined by an with PBS and then stored in RNAlater (Applied Biosystems Inc. end point assay (2). The reaction buffer consisted of 50 mM (ABI)). Embryos from each day were pooled prior to RNA iso- HEPES (pH 8.0), 100 mM KCl, 5 mM MgCl lation. RNA was isolated using TRI reagent (ABI) and treated , 0.4 mM CH -THF, 2 2 40 mM -mercaptoethanol, and either NAD (1 mM)or with TurboDnase (Invitrogen) following the manufacturer’s NADP (6 mM). Potassium phosphate (25 mM) was also instructions. RNA quality was verified by confirmation of the included for the NAD -dependent activity. To determine the presence of 28S and 18S rRNA bands on an agarose gel. cDNA was synthesized using SuperScript III (Invitrogen) and random cofactor specificity of the MTHFD2L enzyme in conditions that more closely resembled physiological conditions, reaction hexamers. buffer contained 50 mM HEPES (pH 8.0), 100 mM KCl, 0.5 mM Real-time PCR—Primers against mouse MTHFD1, MTHFD1L, MgCl ,10mM potassium phosphate, 40 mM -mercaptoetha- MTHFD2, MTHFD2L, eEF2 (eukaryotic translation elongation factor 2), and TBP (TATA-box-binding protein) were designed nol, and different cofactors including NAD (0.2 mM)or NADP (0.05 mM) and varying concentrations of CH -THF. using Primer-BLAST (25) to yield an amplicon between 95 and Sixty l of reaction mixture without CH -THF and 20 lof 125 base pairs long and cross at least one exon-exon junction purified MTHFD2L were mixed, and the enzyme reaction was (see Table 1). Primers were checked individually against plas- lofCH mids containing mouse MTHFD1, MTHFD1L, MTHFD2, and initiated by the addition of 20 -THF followed by incu- bation at 30 °C for 5 min. The reaction was quenched with 200 MTHFD2L to ensure that the primer pairs were specific to the l of 3% perchloric acid, and the plate was read at 350 nm on an target gene. Quantitative real-time PCR was performed using Infinite M200 (Tecan, Männedorf, Switzerland). The path SYBR Green (Qiagen) on an ABI ViiA 7 using a two-step pro- length was corrected using near-infrared measurements (23). gram (50 °C for 2 min and then 95 °C for 10 min followed by 40 CH -THF cyclohydrolase activity was determined by a con- cycles of 95 °C for 15 s and 60 °C for 15 s). The specificity of the tinuous assay (24) in microplate format. The enzyme reaction reaction was verified by melt curve analysis. Relative expression was incubated at 30 °C with MTHFD2L containing 200 mM values from embryos were calculated by normalizing to TBP potassium maleate (pH 7.4), 20 mM -mercaptoethanol, and (26), and values from adult tissues were normalized to eEF2 varying concentrations of CH -THF. The activity was moni- (27). tored by observing the decrease in absorbance of CH -THF at We have shown previously that MTHFD2L is alternatively 355 nm. spliced at exons 2 and 8 (11). To determine the abundance of the splice variants of MTHFD2L containing exon 2 or exon 8, For the determination of kinetic parameters, the initial rate data were fitted to the Michaelis-Menten equation by nonlinear we designed primer pairs that would bind in exons 2 and 3 or in regression using Prism (GraphPad, La Jolla, CA). Inhibition of exons 7 and 8, so that they would produce only a product from NADP -dependent dehydrogenase activity by phosphate ion splice variants containing exon 2 or exon 8, respectively. The expression values for these primer pairs were normalized to the was examined by assays at four fixed phosphate ion concentra- MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15509 MTHFD2L Exhibits Dual Redox Cofactor Specificity expression levels found using primers MTHFD2L F1 and R1, which bind in exons 6 and 7. There is no evidence for alternative splicing of these exons, and they are thus assumed to represent the total amount of MTHFD2L transcript, including all splice variants. In Situ Hybridization of Whole Mouse Embryos—Mouse embryos ranging from E9.5 to 12.5 were hybridized as described previously using digoxigenin-labeled UTP RNA probes (28). Antisense probes were constructed as described previously (28) by in vitro transcription using T7 RNA polymerase to tran- scribe from Riken clone 1110019K23 (29) linearized with BamHI. Sense probes were made by linearizing with XhoI and transcribing with T3 RNA polymerase. Yeast Complementation Assay—Exon 8 was removed from pET22b-rMTHFD2L by overlap extension PCR (30) using the primers rD2L-x8 5/HindIII F (5-ATCTAAGCTTATGGCG- ACGCGGGCCCGT-3), rD2L-x8 5 R(5-TAACAG CTGCA- GCCACTATGATAATCT-3), rD2L-x8 3 F(5-CTGCAGC- TGTTAAGAAGAAGGCCAGC-3), and rD2L-x8 3/BamHI R (V5) (5-TGAT GGATCC A GTAGGTGATATTCTTGGCA- GCCAG-3). Following PCR, rMTHFD2L-x8 was subcloned into YEp24ES (11, 17) using the HindIII and BamHI restriction sites. Plasmids were transformed into the yeast strain MWY4.5 (ser1 ura3–52 trp1 leu2 his4 ade3–30/65 mtd1), and transformants were selected for uracil prototrophy. The in vivo complementation assay was performed in this yeast strain as described previously (11). Preparation of crude yeast lysates and immunoblotting were carried out as described previously (11). The protein concen- tration was determined by BCA assay (Thermo Fisher Scien- tific). NAD -dependent CH -THF dehydrogenase activity in the lysates was assayed with MgCl and potassium phosphate as described above. RESULTS Expression and Purification of Rat MTHFD2L—Our initial characterization of mammalian MTHFD2L relied on the recombinant full-length rat protein expressed in yeast crude extracts. Difficulties in purification, likely because of its tight association with membranes (11), prevented us from conduct- ing a full enzymatic characterization of MTHFD2L in that FIGURE 2. Purified rat MTHFD2L possesses cyclohydrolase activity. A, study. In the present study, we overcame these problems by His -tagged rat MTHFD2L was purified by immobilized nickel affinity chroma- tography as described under “Experimental Procedures.” Lane 1 shows the using several strategies. To increase the yield of recombinant imidazole elution after washing the column at 4 °C. In a second experiment, protein, we replaced the mitochondrial targeting sequence of the column was washed at room temperature instead of 4 °C. Lane 2 shows the room temperature wash fractions. Lane 3 shows the imidazole elution the rat MTHFD2L with an N-terminal His tag for localization after washing at room temperature. B, dependence of cyclohydrolase activity in the yeast cytosol (31). Despite the loss of its targeting on (6R,S)5,10-methenyl-THF concentration. Results shown are the means sequence, the protein remained associated with cellular mem- S.E. of five replicates (error bars are included for all data points but are obscured by the data symbol when the scatter is small). branes; so we included sodium carbonate in the extraction buffer to release MTHFD2L from membranes (32). Finally, we washed the Ni column at room temperature rather than at The specific activity shown in Fig. 2B is 11-fold higher than 4 °C to eliminate contamination from endogenous yeast pro- the buffer-catalyzed rate. Cyclohydrolase activity could not be teins (33). This washing step allowed the purification of determined at saturating conditions due to the high absorbance MTHFD2L to greater than 95% homogeneity (Fig. 2A, cf. lanes of substrate, and thus accurate values for k and K could not cat m 1 and 3). The mobility of the purified protein is consistent with be calculated. the expected size of 33 KDa. Steady-state Kinetics and Cofactor Specificity of 5,10-Methyl- MTHFD2L Possesses 5,10-Methenyl-THF Cyclohydrolase ene-THF Dehydrogenase Activity—We reported previously that Activity—MTHFD2L exhibits robust CH -THF cyclohydro- recombinant full-length rat MTHFD2L expressed in yeast exhibits NADP -dependent CH -THF dehydrogenase activity lase activity, confirming the bifunctional nature of this enzyme. 15510 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity TABLE 2 Kinetic parameters for MTHFD2L 5,10-methylene-THF dehydrogen- ase activity Enzyme assays were performed as described under “Experimental Procedures.” CH -THF kinetic parameters were determined using saturating concentrations of NAD (1.0 mM) or NADP (6.0 mM). When NAD was used, potassium phosphate (25 mM) and MgCl (5 mM) were also included. Redox cofactor kinetic parameters were determined using saturating concentrations of CH -H PteGlu (400 M). 2 4 1 Substrate NAD /NADP CH THF k k /K 2 cat cat m 1 1 1 M s s M NAD 147  16 3.6  0.1 0.025 CH -H PteGlu 40  5 2.7  0.07 0.067 2 4 1 CH -H PteGlu 130  30 8.8  0.4 0.068 2 4 5 NADP 537  54 1.1  0.04 0.002 CH -H PteGlu 42  7 1.3  0.05 0.030 2 4 1 CH -H PteGlu 153  39 7.2  0.5 0.047 2 4 5 in crude extracts (11). Purified MTHFD2L exhibited both NAD - and NADP -dependent CH -THF dehydrogenase activity using either mono- or pentaglutamylated CH -THF (CH -H PteGlu and CH -H PteGlu , respectively). The 2 4 1 2 4 5 kinetic parameters are given in Table 2. Values for k and K cat m differed between the polyglutamylation states of the CH -THF substrate, but they were not sensitive to the redox cofactor in the dehydrogenase reaction (NAD or NADP ). The K values for CH -H PteGlu were 3.5 times higher than those of CH - 2 4 5 2 H PteGlu regardless of the redox cofactor. However, this was 4 1 accompanied by a similar increase in k , resulting in similar cat k /K values (Table 2). Using CH -H PteGlu , the K values cat m 2 4 1 m for NAD and NADP were 147  16 and 537  54 M, respectively (Table 2). Although the dehydrogenase activity of the bifunctional MTHFD2 (an isozyme of MTHFD2L) is classified as NAD - FIGURE 3. 5,10-Methylene-THF dehydrogenase activity of MTHFD2L. A, specific, it can also utilize NADP at a reduced efficiency (6). purified MTHFD2L was assayed for NAD - and NADP -dependent 5,10-methyl- The NAD -dependent activity of MTHFD2 requires Mg and 2 ene-THF dehydrogenase activity in the presence or absence of P and Mg . Enzyme activity is expressed as mol product/min/mg of protein. Each column inorganic phosphate (P ) (6). To explore redox cofactor speci- represents the mean  S.E. of triplicate determinations. Reaction buffer con- ficity in MTHFD2L, we measured specific activity using satu- tained 50 mM HEPES (pH 8.0), 100 mM KCl, 0.4 mM 5,10-CH -THF, and 40 mM rating levels of NAD or NADP in the presence of Mg -mercaptoethanol. NAD was used at 1.0 mM and NADP at 6.0 mM.Mg and were tested by including 5.0 mM MgCl and 25 mM potassium phosphate and/or P (Fig. 3A). When NAD was used, the dehydrogenase i 2 where indicated. B, inhibition of NADP -dependent 5,10-methylene-THF dehy- activity of MTHFD2L was strongly dependent on the presence drogenase activity by inorganic phosphate. Each point represents the mean of Mg and P in combination. Using CH -H PteGlu and S.E. of triplicate determinations (error bars are included for all data points but are i 2 4 1 obscured by the data symbol when the scatter is small). The curves represent NAD , the K values for Mg and P were 233 62 and 293 m i nonlinear fits to the Michaelis-Menten model. The reaction buffer (as in A) con- 59 M, respectively. When NADP was used, MTHFD2L activ- tained 5.0 mM MgCl and varying concentrations of NADP . Potassium phos- phate concentrations were 0 (●),5(), 10 (Œ), and 25 (ƒ)mM. ity was increased slightly with Mg . Inorganic phosphate appeared to counteract the Mg effect with NADP (Fig. 3A). Because MTHFD2 shows inhibition of NADP -dependent see the legend to Fig. 4 for references). For each component, the dehydrogenase activity by inorganic phosphate (6), we sought ratio of NAD -dependent to NADP -dependent dehydrogen- to determine if MTHFD2L behaves in the same way. We ase activities was plotted (Fig. 4). The data indicate that there is observed that increasing concentrations of phosphate ion an 3.5-fold preference for NAD at physiological P concen- reduced the NADP -dependent dehydrogenase activity of trations (Fig. 4A) and a 5–6-fold preference for NAD at phys- MTHFD2L (Fig. 3B). The data fit best to a competitive inhibi- iological Mg concentrations (Fig. 4B). At high concentra- tion model with a K of 1.9  0.3 mM. tions of CH -THF, MTHFD2L clearly prefers NAD (Fig. 4C). i 2 Cofactor Specificity at Physiological Concentrations of Mg , However, as the CH -THF concentration is lowered to more P , and CH -THF—To better understand the cofactor prefer- physiological levels, the ratio approaches 1, and at the lowest i 2 ence (NAD versus NADP ) of the MTHFD2L dehydrogenase CH -THF concentrations, the enzyme is more active with activity under physiologically relevant substrate conditions, we NADP . This suggests that cofactor preference will be very repeated the assay at a wide range of P ,Mg , and CH -THF sensitive to CH -THF concentration in vivo. i 2 2 concentrations. The range used includes concentrations esti- Gene Expression Profile of MTHFD Gene Family in Mouse mated to exist in the matrix compartment of mammalian mito- Embryos—It has been reported previously that the MTHFD2 chondria (CH gene is expressed in early embryos, but its expression declines THF 2.5–25 M,Mg 0.5 mM,P 10 mM; 2- i MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15511 MTHFD2L Exhibits Dual Redox Cofactor Specificity FIGURE 5. Temporal expression profile of MTHFD gene family in mouse embryos. The relative expression profiles of MTHFD1 (●), MTHFD1L (f), MTHFD2 (Œ), and MTHFD2L () was determined by real-time PCR as described under “Experimental Procedures.” The age of the embryos from which the RNA was obtained is indicated in embryonic days (birth occurs at E20.0). mRNA expression was normalized to that of the TBP transcript. Each point represents the mean  S.E. of triplicate determinations (error bars are included for all data points but are obscured by the data symbol when the scatter is small). as the embryo approaches birth, and the MTHFD2 enzyme is found only in nondifferentiated tissues in adults (9, 28). We determined the relative expression profiles of all four MTHFD gene family members in mouse embryos using real-time PCR, normalizing to TBP (TATA-box-binding protein) expression. TBP has been determined to be stably expressed at multiple stages of embryonic development, making it a suitable house- keeping gene for real-time PCR experiments involving embryos (26). MTHFD1 and MTHFD1L transcript expression was high- est at E8.5 and subsequently decreased until expression increased again at E13.5–17.5 (Fig. 5). MTHFD2 expression was low at all embryonic days examined. MTHFD2L was also expressed at all embryonic days examined. MTHFD2L expres- sion was low at E8.5 but increased beginning at E10.5 (Fig. 5). These profiles suggest that a switch between MTHFD2 and MTHFD2L expression occurs approximately between embry- onic days 8.5 and 10.5 during mouse embryogenesis. In situ hybridization of whole mouse embryos (Fig. 6) revealed that MTHFD2L is expressed in the neural tube and the forebrain, midbrain, and hindbrain, suggesting a possible role in neural tube development. Other areas of intense staining included the branchial arches and limb buds, particularly along the progress zone. Gene Expression Profile of MTHFD Gene Family in Adult Mice—Expression of adult MTHFD genes from male and female mice was normalized to the expression of eEF2, a house- keeping gene that is stably expressed in a wide array of adult tissues (27). The MTHFD2L transcript was expressed in all of the tissues examined, with the highest expression observed in FIGURE 4. Redox cofactor specificity of MTHFD2L at physiological con- centrations of P ,Mg , and 5,10-CH -THF. The ratio of NAD - to NADP - i 2 dependent 5,10-methylene-THF activity was plotted as a function of increas- dent reactions. For the NADP -dependent reactions, only 5 mM MgCl was ing concentrations of inorganic phosphate (A), Mg (B), or 5,10-CH -THF (C). included. Reported mitochondrial matrix substrate concentration ranges Reaction buffer (described in the legend for Fig. 3) contained either 1 mM (indicated by shaded areas) are 5–15 mM for P (47– 49), 0.3– 0.4 mM for Mg NAD or6mM NADP . 5,10-CH -THF was included at 0.4 mM (A and B)or (48, 49), and 2.5–25 M for 5,10-CH THF (37– 40), respectively. The best esti- 2 2 varied (C). For dependence on P mates found for in vivo mitochondrial NADP concentration (A),5mM MgCl was included and NAD concentrations are i 2 for both NAD - and NADP -dependent reactions. For dependence on Mg NADP 80 M and NAD 240 M (42, 43). The data in C were fit to the concentration (B), 25 mM potassium phosphate was included in NAD Michaelis-Menten equation. The 5,10-CH -de- -THF concentration that gave the pendent but not NADP -dependent reactions. For dependence on 5,10-CH - half-maximal ratio of NAD - to NADP -dependent 5,10-methylene-THF THF concentration, 5 mM MgCl activity was 12.5 M. and 25 mM P were included in NAD -depen- 2 i 15512 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity FIGURE 6. Spatial distribution of MTHFD2L expression in mouse embryos. In situ hybridizations of whole mount embryos ranging from E9.5 to E12.5 (A–D) were performed using digoxigenin-labeled UTP RNA probes as described under “Experimental Procedures.” C, as shown on the E11.5 embryo, MTHFD2L is especially prominent in the neural tube (a), forebrain (b), midbrain (c), hindbrain (d), branchial arches (e), and somites (f). Embryos A–C were imaged at the same magnification (20). Embryo D was imaged at 12.5 magnification. Scale bars correspond to 1 mm. FIGURE 8. MTFHD2L splice variants during mouse development. The rel- ative abundance of exon 2- (f) and exon 8-containing (●) MTHFD2L tran- scripts was determined by real-time PCR and analyzed as described under “Experimental Procedures.” Each point represents the mean  S.E. of tripli- cate determinations (‘bars are included for all data points but are obscured by the data symbol when the scatter is small). Alternative Splicing of MTHFD2L—We have shown previ- ously that MTHFD2L is alternatively spliced at exons 2 and 8 (11), with either exon skipped or included in the mature mRNA. The skipping of exon 2 and inclusion of exon 8 produces an enzymatically active enzyme of 347 residues (including the 40-residue mitochondrial targeting sequence) (11). The inclu- sion of exon 2 results in the introduction of an early stop codon. However, translation beginning from an ATG in exon 3 is pre- dicted to produce a truncated protein missing the mitochon- drial targeting sequence. When exon 8 is skipped, exon 7 is spliced to exon 9 with the reading frame intact, resulting in FIGURE 7. Expression profile of MTHFD gene family in adult mouse tis- deletion of 43 codons. Using total embryo RNA from embryos sues. The relative expression profiles of MTHFD1 (green), MTHFD1L (red), ages E8.5 to E17.5, we observed that exon 2 was present in less MTHFD2 (blue), and MTHFD2L (black) was determined by real-time PCR as than 10% of the transcripts in all of the embryonic days exam- described under “Experimental Procedures.” mRNA expression was normal- ized to that of the eEF2 transcript in female (top) and male (bottom) adult ined (Fig. 8), indicating that the vast majority of MTHFD2L mice. Each column represents the mean  S.E. of triplicate determinations. transcripts encode a mitochondrial targeting sequence. Exon 8, brain and lung and lower expression in liver and kidney (Fig. 7). on the other hand, was found to be missing in 20–45% of the As has been reported previously, MTHFD1 showed the highest MTHFD2L transcripts throughout embryogenesis (Fig. 8). expression in liver and kidney (34). MTHFD1L was most highly To determine what effect the removal of exon 8 would have expressed in brain and spleen. The MTHFD2 transcript could on MTHFD2L activity, MTHFD2L lacking exon 8 was be detected at low levels in most tissues, but only testis and expressed in yeast. NAD -dependent CH -THF dehydroge- spleen expressed it at even a modest level. The expression pat- nase activity could not be detected in crude yeast lysate from terns of the four transcripts were similar in males and females. yeast cells transformed with the truncated construct (YEp- MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15513 MTHFD2L Exhibits Dual Redox Cofactor Specificity sesses both CH -THF dehydrogenase and CH -THF cyclohy- drolase activities (Fig. 2). The dehydrogenase activity of this bifunctional enzyme can use either NAD or NADP but requires both phosphate and Mg when using NAD (Fig. 3A). The NADP -dependent dehydrogenase activity of MTHFD2L is inhibited by inorganic phosphate (Fig. 3B). MTHFD2L can use the mono- and polyglutamylated forms of CH -THF with similar catalytic efficiencies (k /K ; Table 2). 2 cat m Expression of the MTHFD2L transcript is low in early mouse embryos, begins to increase at E10.5, and continues through birth (Fig. 5). In adults, MTHFD2L was expressed in all tissues examined, with the highest levels observed in brain and lung (Fig. 7). How do these cofactor requirements compare with those of the other CH -THF dehydrogenase/CH -THF cyclohydrolase found in mammalian mitochondria? The MTFHD2 isozyme FIGURE 9. Expression of MTHFD2L lacking exon 8 in yeast. S. cerevisiae strain has been named NAD -dependent methylenetetrahydrofolate MWY4.5 (ser1 ura3 trp1 leu2 his4 ade3–30/65 mtd1) was transformed to uracil dehydrogenase-methenyltetrahydrofolate cyclohydrolase (6, prototrophy with YEp-rD2L (wild type), YEp-rD2L-x8 (lacking exon 8), or empty 10) but in fact exhibits dehydrogenase activity with NADP , vector (YEp24ES). Ura transformants were streaked onto yeast minimal plates containing serine as a one-carbon donor plus adenine (left) or serine alone (right) albeit with a much higher K and lower V (6). In fact, the m max and incubated at 30 °C for 4 days. Both plates also contained leucine, tryptophan, redox cofactor requirements of the two isozymes are quite sim- and histidine to support the other auxotrophic requirements of MWY4.5. Inset, ilar: both exhibit lower K values for NAD than for NADP ; immunoblot of whole cell lysate from MWY4.5 transformed with the indicated plasmids. Each lane was loaded with 50 g of protein. The blot was probed with their NAD -dependent activities require phosphate and Mg ; polyclonal antibodies against MTHFD2L (1:1000 dilution). and their NADP -dependent activities are inhibited by phos- phate. The absolute requirement of the NAD -dependent rD2L-x8; data not shown). NAD -dependent CH -THF dehy- activity of MTHFD2 for Mg and P has been characterized in 2 i great detail by Mackenzie and co-workers (10). MTHFD2 uses drogenase activity was easily detectable in lysate from yeast cells transformed with the full-length construct. Mg and P to convert an NADP binding site into an NAD We next asked whether MTHFD2L lacking exon 8 was active binding site. P binds in close proximity to the 2-hydroxyl of in vivo, using a yeast complementation assay (11). Briefly, this NAD and competes with NADP binding. Mg plays a role in positioning P and NAD. Mackenzie and co-workers (10) iden- assay uses yeast strain MWY4.5, which lacks cytoplasmic CH - THF dehydrogenase activities as well as the 10-formyl-THF tified several amino acid residues in MTHFD2 that are involved synthetase activity of the cytoplasmic trifunctional C -THF in the P and Mg binding, and these residues are highly con- 1 i synthase (35). Wild-type yeast can produce 10-CHO-THF for served in MTHFD2L in mammals. It is thus likely that Mg and P play a mechanistically similar role in the NAD -depen- de novo purine biosynthesis from serine using either cytoplas- mic or mitochondrial 1C pathways (36) (see Fig. 1). However, dent dehydrogenase activity of MTHFD2L as well. MWY4.5 is blocked in both pathways to 10-CHO-THF. This At saturating levels of CH -THF, using NAD as a cofactor, blockage creates a requirement for adenine in the growth the k /K value for the CH -THF dehydrogenase reaction of cat m 2 1 1 MTHFD2 is 2.9 s M (8). This value is 45-fold higher medium (35). Cytoplasmically localized MTHFD2L lacking exon 8 should rescue the adenine requirement of MWY4.5 if it is cata- than the efficiency exhibited by MTHFD2L (k /K 0.067 cat m 1 1 lytically active in vivo. Transformants of MWY4.5 harboring YEp- s M ). These differences in kinetic parameters are unlikely rD2L-x8, YEp-rD2L (full-length MTHFD2L) (11), or empty vector to be due to different assay conditions. When we performed the CH -THF dehydrogenase assay under buffer conditions that (YEp24ES) were streaked onto yeast minimal plates containing serine as a one-carbon donor or serine adenine and incubated at were used to characterize MTHFD2 (MOPS (pH 7.3)) rather 30 °C. As shown in Fig. 9, full-length MTHFD2L, expressed from than the HEPES (pH 8.0) used in this article, we did not observe significant changes in the values for k YEp-rD2L as a positive control, fully complemented the adenine or K (data not shown). cat m The apparent preference of MTHFD2L for NAD (Fig. 3A)is requirement of MWY4.5, as observed previously (11). However, MTHFD2L lacking exon 8, expressed from the rD2L-x8 plasmid, most dramatic at nonphysiological levels of phosphate, Mg , did not complement the adenine requirement. An immunoblot of and folate cofactor. When these experiments were repeated crude yeast lysates from these transformants confirmed that the at more physiologically relevant substrate concentrations, MTHFD2L showed much less preference for NAD (Fig. 4). In truncated protein was expressed at a level similar to that of the full-length protein in control cells (Fig. 9, inset). These in vitro and fact, at CH -THF concentrations below 10 M, the enzyme is in vivo results suggest that the MTHFD2L variant lacking exon 8 more active with NADP than with NAD (Fig. 4C). Given that does not function as a CH -THF dehydrogenase. estimates for mitochondrial matrix levels of CH -THF range 2 2 from 2.5 to 25 M (37–40), it is likely that MTHFD2L exhibits DISUSSION dual redox cofactor specificity in vivo. The experiments described here demonstrate that the mam- The use of NAD versus NADP in this step can have a malian MTHFD2L isozyme, like MTHFD1 and MTHFD2, pos- dramatic effect on the rate and direction of flux of one-carbon 15514 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity units through this pathway in mitochondria. The oxidation tion for gluconeogenesis. When CH -THF dehydrogenase and state of mitochondrial pools of NAD and NADP is dictated CH -THF cyclohydrolase activities are included in the model, by mitochondrial respiration (41), which in turn is linked to the mitochondrial folate pathway produces formate for cyto- nutrition, differentiation, and proliferation (42). Measurements solic export, where it is incorporated into purines, thymidylate, in liver suggest that the redox potential of the NAD /NADH and the methyl cycle (37). Christensen and MacKenzie (44) have proposed that the level of MTHFD2 expression could act matrix pool is typically 75–100 mV more positive than that of the NADP /NADPH matrix pool (41, 43). Thus, the ratio of as a metabolic switch to control the balance between serine and CH -THF to 10-CHO-THF in the matrix (reactions 3m and 2m formate production. in Fig. 1) will be shifted much further toward 10-CHO-THF We suggest that the existence of two mitochondrial dehydro- genase/cyclohydrolase isozymes in mammals (MTHFD2 and with a CH -THF dehydrogenase linked to the NAD /NADH pool versus the NADP /NADPH pool (6, 7). A cyclohydrolase/ MTHFD2L) reflects the need to tightly regulate flux through dehydrogenase with dual cofactor specificity, such as this oxidation step in response to changing metabolic condi- MTHFD2L, would be able to adapt immediately to changing tions and needs. For example, de novo purine biosynthesis is metabolic conditions, shifting the equilibrium between CH especially important in rapidly dividing cells, such as during THF and 10-CHO-THF (and formate) depending on the rela- embryogenesis. Thus, early embryos express both MTHFD2 tive levels of oxidized cofactor (NAD or NADP ) in the mito- and MTHFD2L isozymes, ensuring that mitochondrial formate chondrial matrix. We do not know whether the MTHFD2 production is adequate to support de novo purine biosynthesis. Indeed, embryonic growth and neural tube closure requires isozyme might also exhibit dual redox cofactor specificity in vivo, as it was not characterized at physiologically relevant sub- mitochondrial formate production (45). Compared with strate concentrations (6, 10). embryos, however, adult mammals do not have a high demand We determined expression profiles for the entire MTHFD for de novo purine biosynthesis (46). The loss of expression of family of genes during mouse embryogenesis (Fig. 5) and in MTHFD2 as the embryos approach birth may reflect the lower adult tissues (Fig. 7). The results for MTHFD1 (cytoplasmic demand for de novo purine biosynthesis in neonate and adult reactions 1–3 (Fig. 1)) and MTHFD1L (mitochondrial reaction mammals. 1m) are qualitatively similar to previously reported transcript In addition to switching between expressing one or two expression patterns in mouse embryos based on a staged mitochondrial CH -THF dehydrogenase/CH -THF cyclohy- Northern blot (28). Both transcripts are highest in early drolase enzymes, there may be other ways of regulating embryos and decrease during embryonic days 9.5–15.5, only to MTHFD2L expression such as alternative splicing. We have increase again as the embryo approaches birth. MTHFD2 and shown previously that an exon 8 deletion is present in adult MTHFD2L, on the other hand, exhibit very different expression tissues (11). We observed significant expression of this same profiles. MTHFD2 expression was low in all embryonic days splice variant of MTHFD2L throughout embryogenesis (Fig. 8). examined, whereas expression of the MTHFD2L transcript However the protein that would be produced from the tran- increased beginning at E10.5 and remained elevated through script lacking exon 8 did not show activity in vitro or in vivo (Fig. birth (Fig. 5). These data reveal a switch from MTHFD2 to 9), and the function of this splice variant is unknown. MTHFD2L expression at about the time of neural tube closure in mouse embryos. The spatial expression of MTHFD2L is Acknowledgments—We thank Nafee Talukder for assistance with the localized to the neural tube, developing brain, branchial arches, purification of recombinant MTHFD2L and Jordan Lewandowski for and limb buds (Fig. 6). These regions are also areas where performing the in situ hybridizations. MTHFD2 and MTHFD1L are expressed (28), suggesting a role for the mitochondrial folate pathway in these embryonic REFERENCES tissues. 1. Tibbetts, A. S., and Appling, D. R. (2010) Compartmentalization of mam- Why might mammals possess these two distinct mitochon- malian folate-mediated one-carbon metabolism. Annu. Rev. Nutr. 30, drial dehydrogenase/cyclohydrolase isozymes? It appears that 57–81 under most conditions, the majority of 1C units for cytoplasmic 2. Walkup, A. S., and Appling, D. R. (2005) Enzymatic characterization of processes are derived from mitochondrial formate (1). Oxida- human mitochondrial C1-tetrahydrofolate synthase. Arch. Biochem. Bio- tion of mitochondrial CH -THF is essential for the production phys. 442, 196–205 3. Mejia, N. R., and MacKenzie, R. E. (1985) NAD-dependent methylenetet- of 10-CHO-THF, which is processed by MTHFD1L to provide rahydrofolate dehydrogenase is expressed by immortal cells. J. Biol. Chem. formate for cytoplasmic export (Fig. 1). This formate is then 260, 14616–14620 reattached to THF for use in de novo purine biosynthesis or 4. Mejia, N. R., Rios-Orlandi, E. M., and MacKenzie, R. E. (1986) NAD-de- further reduced for either thymidylate synthesis or remethyla- pendent methylenetetrahydrofolate dehydrogenase-methenyltetrahydro- tion of homocysteine to methionine. Modeling studies suggest folate cyclohydrolase from ascites tumor cells: purification and properties. that the oxidation step (reaction 3m (Fig. 1)), catalyzed by J. Biol. Chem. 261, 9509–9513 5. Mejia, N. R., and MacKenzie, R. E. (1988) NAD-dependent methylenetet- either MTHFD2 or MTHFD2L, is a critical control point for rahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase mitochondrial 1C metabolism. Using an in silico model, in transformed cells is a mitochondrial enzyme. Biochem. Biophys. Res. Nijhout et al. (37) observed that the exclusion of CH -THF Comm. 155, 1–6 dehydrogenase and CH -THF cyclohydrolase activities from 6. Yang, X. M., and MacKenzie, R. E. (1993) NAD-dependent methylenetet- the mitochondrial folate pathway results in loss of formate rahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the export and a dramatic increase in mitochondrial serine produc- MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15515 MTHFD2L Exhibits Dual Redox Cofactor Specificity yeast MIS1 gene. Biochemistry 32, 11118–11123 28. Pike, S. T., Rajendra, R., Artzt, K., and Appling, D. R. (2010) Mitochondrial 7. Pelletier, J. N., and MacKenzie, R. E. (1995) Binding and interconversion of C1-THF synthase (MTHFD1L) supports flow of mitochondrial one-car- tetrahydrofolates at a single site in the bifunctional methylenetetrahydro- bon units into the methyl cycle in embryos. J. Biol. Chem. 285, 4612–4620 folate dehydrogenase/cyclohydrolase. Biochemistry 34, 12673–12680 29. Kawai, J., Shinagawa, A., Shibata, K., Yoshinom M., Itoh, M., Ishii, Y., 8. Pawelek, P. D., and MacKenzie, R. E. (1998) Methenyltetrahydrofolate Arakawa, T., Hara, A., Fukunishi, Y., Konno, H., Adachi, J., Fukuda, S., cyclohydrolase is rate limiting for the enzymatic conversion of 10-form- Aizawa, K., Izawa, M., Nishi, K., Kiyosawa, H., Kondo, S., Yamanaka, I., yltetrahydrofolate to 5,10- methylenetetrahydrofolate in bifunctional de- Saito, T., Okazaki, Y., Gojobori, T., Bono, H., Kasukawa, T., Saito, R., hydrogenase-cyclohydrolase enzymes. Biochemistry 37, 1109–1115 Kadota, K., Matsuda, H., Ashburner, M., Batalov, S., Casavant, T., Fleis- 9. Di Pietro, E., Wang, X. L., and MacKenzie, R. E. (2004) The expression of chmann, W., Gaasterland, T., Gissi, C., King, B., Kochiwa, H., Kuehl, P., mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase Lewis, S., Matsuo, Y., Nikaido, I., Pesole, G., Quackenbush, J., Schriml, supports a role in rapid cell growth. Biochim. Biophys. Acta 1674, 78–84 L. M., Staubli, F., Suzuki, R., Tomita, M., Wagner, L., Washio, T., Sakai, K., 10. Christensen, K. E., Mirza, I. A., Berghuis, A. M., and Mackenzie, R. E. Okido, T., Furuno, M., Aono, H., Baldarelli, R., Barsh, G., Blake, J., Boffelli, (2005) Magnesium and phosphate ions enable NAD binding to methyl- D., Bojunga, N., Carninci, P., de Bonaldo, M. F., Brownstein, M. J., Bult, C., enetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohy- Fletcher, C., Fujita, M., Gariboldi, M., Gustincich, S., Hill, D., Hofmann, drolase. J. Biol. Chem. 280, 34316–34323 M., Hume, D. A., Kamiya, M., Lee, N. H., Lyons, P., Marchionni, L., 11. Bolusani, S., Young, B. A., Cole, N. A., Tibbetts, A. S., Momb, J., Bryant, Mashima, J., Mazzarelli, J., Mombaerts, P., Nordone, P., Ring, B., Ringwald, J. D., Solmonson, A., and Appling, D. R. (2011) Mammalian MTHFD2L M., Rodriguez, I., Sakamoto, N., Sasaki, H., Sato, K., Schönbach, C., Seya, Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase T., Shibata, Y., Storch, K. F., Suzuki, H., Toyo-oka, K., Wang, K. H., Weitz, isozyme expressed in adult tissues. J. Biol. Chem. 286, 5166–5174 C., Whittaker, C., Wilming, L., Wynshaw-Boris, A., Yoshida, K., Hase- 12. Blakley, R. L. (1957) The interconversion of serine and glycine: prepara- gawa, Y., Kawaji, H., Kohtsuki, S., Hayashizaki, Y, RIKEN Genome Explo- tion and properties of catalytic derivatives of pteroylglutamic acid. ration Research Group Phase II Team, and the FANTOM Consortium Biochem. J. 65, 331–342 (2001) Functional annotation of a full-length mouse cDNA collection. 13. Curthoys, N. P., and Rabinowitz, J. C. (1971) Formyltetrahydrofolate syn- Nature 409, 685–690 thetase: binding of adenosine triphosphate and related ligands determined 30. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J.K., and Pease, L. R. (1989) by partition equilibrium. J. Biol. Chem. 246, 6942–6952 Site-directed mutagenesis by overlap extension using the polymerase 14. Appling, D. R., and West, M. G. (1997) Monofunctional NAD-dependent, chain reaction. Gene. 77, 51–59 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces 31. Zhang, L., Joshi, A. K., and Smith, S. (2003) Cloning, expression, charac- cerevisiae. Methods Enzymol. 281, 178–188 terization, and interaction of two components of a human mitochondrial 15. Kallen, R. G., and Jencks, W. P. (1966) The mechanism of the condensa- fatty acid synthase: malonyltransferase and acyl carrier protein. J. Biol. tion of formaldehyde with tetrahydrofolic acid. J. Biol. Chem. 241, Chem. 278, 40067–40074 5851–5863 32. Prasannan, P., and Appling, D. R. (2009) Human mitochondrial C1-tetra- 16. Rabinowitz, J. C. (1963) Preparation and properties of 5,10-methenyltet- hydrofolate synthase: submitochondrial localization of the full-length en- rahydrofolic acid and 10-formyltetrahydrofolic acid. Methods Enzymol. 6, zyme and characterization of a short isoform. Arch. Biochem. Biophys. 814–815 481, 86–93 17. Suliman, H. S., Sawyer, G. M., Appling, D. R., and Robertus, J. D. (2005) 33. Dihazi, H., Kessler, R., and Eschrich, K. (2001) One-step purification of Purification and properties of cobalamin-independent methionine syn- recombinant yeast 6-phosphofructo-2-kinase after the identification of thase from Candida albicans and Saccharomyces cerevisiae. Arch. contaminants by MALDI-TOF MS. Protein Expr. Purif. 21, 201–209 Biochem. Biophys. 441, 56–63 34. Thigpen, A. E., West, M. G., and Appling, D. R. (1990) Rat C -tetrahydro- 18. Claros, M. G., and Vincens, P. (1996) Computational method to predict folate synthase: cDNA isolation, tissue-specific levels of the mRNA, and mitochondrially imported proteins and their targeting sequences. Eur. expression of the protein in yeast. J. Biol. Chem. 265, 7907–7913 J. Biochem. 241, 779–786 35. West, M. G., Horne, D. W., and Appling, D. R. (1996) Metabolic role of 19. Li, M. Z., and Elledge, S. J. (2007) Harnessing homologous recombination cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in vitro to generate recombinant DNA via SLIC. Nat. Methods 4, 251–256 in Saccharomyces cerevisiae. Biochemistry 35, 3122–3132 20. West, M. G., Barlowe, C. K., and Appling, D. R. (1993) Cloning and char- 36. Barlowe, C. K., and Appling, D. R. (1990) Molecular genetic analysis of acterization of the Saccharomyces cerevisiae gene encoding NAD-depen- Saccharomyces cerevisiae C -tetrahydrofolate synthase mutants reveals a dent 5,10-methylenetetrahydrofolate dehydrogenase. J. Biol. Chem. 268, noncatalytic function of the ADE3 gene product and an additional folate- 153–160 dependent enzyme. Mol. Cell. Biol. 10, 5679–5687 21. Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium 37. Nijhout, H. F., Reed, M. C., Lam, S. L., Shane, B., Gregory, J. F., 3rd, and acetate/single-stranded carrier DNA/polyethylene glycol method. Meth- Ulrich, C. M. (2006) In silico experimentation with a model of hepatic ods Enzymol. 350, 87–96 mitochondrial folate metabolism. Theor. Biol. Med. Model. 3, 40 22. Wagner, W., Breksa, A. P., 3rd, Monzingo, A. F., Appling, D. R., and Rober- 38. Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. tus, J. D. (2005) Kinetic and structural analysis of active site mutants of (1989) Folate-pool interconversions and inhibition of biosynthetic pro- monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehy- cesses after exposure of L1210 leukemia cells to antifolates: experimental drogenase from Saccharomyces cerevisiae. Biochemistry 44, 13163–13171 and network thermodynamic analyses of the role of dihydrofolate poly- 23. Palmer, K. F., and Williams, D. (1974) Optical properties of water in the glutamates in antifolate action in cells. J. Biol. Chem. 264, 17016–17023 near infrared. J. Opt. Soc. Am. 64, 1107–1110 39. Horne, D. W., Patterson, D., and Cook, R. (1989) Effect of nitrous oxide 24. Barlowe, C. K., and Appling, D. R. (1990) Isolation and characterization of inactivation of vitamin B -dependent methionine synthetase on the sub- a novel eukaryotic monofunctional NAD -dependent 5,10-methylenetet- cellular distribution of folate coenzymes in rat liver. Arch. Biochem. Bio- rahydrofolate dehydrogenase. Biochemistry 29, 7089–7094 phys. 270, 729–733 25. Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, 40. Eto, I., and Krumdieck, C. L. (1982) Determination of three different pools T. L. (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134 of reduced one-carbon-substituted folates. III. Reversed-phase high-per- 26. Willems, E., Mateizel, I., Kemp, C., Cauffman, G., Sermon, K., and Leyns, formance liquid chromatography of the azo dye derivatives of p-amino- L. (2006) Selection of reference genes in mouse embryos and in differen- benzoylpoly--glutamates and its application to the study of unlabeled tiating human and mouse ES cells. Int. J. Dev. Biol. 50, 627–635 endogenous pteroylpolyglutamates of rat liver. Anal. Biochem. 120, 27. Kouadjo, K. E., Nishida, Y., Cadrin-Girard, J. F., Yoshioka, M., and St- 323–329 Amand, J. (2007) Housekeeping and tissue-specific genes in mouse tissues. 41. Veech, R. L. (1987) Pyridine nucleotides and control of metabolic pro- BMC Genomics 8, 127 cesses, in Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and 15516 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity Medical Aspects, Part B (Dolphin, D., Poulson, R., and Avramovic, O., eds) lethality and neural tube and craniofacial defects in mice. Proc. Natl. Acad. pp. 79–105, John Wiley & Sons, New York Sci. U.S.A. 110, 549–554 42. Yang, H., Yang, T., Baur, J. A., Perez, E., Matsui, T., Carmona, J. J., Lam- 46. Alexiou, M., and Leese, H. J. (1992) Purine utilisation, de novo synthesis ming, D. W., Souza-Pinto, N. C., Bohr, V. A., Rosenzweig, A., de Cabo, R., and degradation in mouse preimplantation embryos. Development 114, Sauve, A. A., and Sinclair, D. A. (2007) Nutrient-sensitive mitochondrial 185–192 NAD levels dictate cell survival. Cell 130, 1095–1107 47. Albe, K. R., Butler, M. H., and Wright, B. E. (1990) Cellular concentrations 43. Sies, H. (1982) Nicotinamide nucleotide compartmentation, in Metabolic of enzymes and their substrates. J. Theor. Biol. 143, 163–195 Compartmentation (Sies, H., ed) pp. 205–231, Academic Press, New York 48. Corkey, B. E., Duszynski, J., Rich, T. L., Matschinsky, B., and Williamson, 44. Christensen, K. E., and MacKenzie, R. E. (2006) Mitochondrial one-car- J. R. (1986) Regulation of free and bound magnesium in rat hepatocytes bon metabolism is adapted to the specific needs of yeast, plants and mam- and isolated mitochondria. J. Biol. Chem. 261, 2567–2574 mals. Bioessays 28, 595–605 49. Saleet Jafri, M., and Kotulska, M. (2006) Modeling the mechanism of met- 45. Momb, J., Lewandowski, J. P., Bryant, J. D., Fitch, R., Surman, D. R., Vokes, abolic oscillations in ischemic cardiac myocytes. J. Theor. Biol. 242, S. A., and Appling, D. R. (2013) Deletion of Mthfd1l causes embryonic 801–817 MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15517 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues *

Download PDF
11 pages

Loading next page...
 
/lp/american-society-for-biochemistry-and-molecular-biology/mitochondrial-mthfd2l-is-a-dual-redox-cofactor-specific-VTeT3yjotg

References (53)

Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2014 Elsevier Inc.
ISSN
0021-9258
eISSN
1083-351X
DOI
10.1074/jbc.m114.555573
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 22, pp. 15507–15517, May 30, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues Received for publication, February 3, 2014, and in revised form, April 14, 2014 Published, JBC Papers in Press, April 14, 2013, DOI 10.1074/jbc.M114.555573 Minhye Shin, Joshua D. Bryant, Jessica Momb, and Dean R. Appling From the Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712 Background: Mitochondria produce one-carbon units for cytoplasmic nucleotide and methyl group synthesis. Results: MTHFD2L uses both NAD and NADP and is expressed in embryonic tissues during neural tube closure. Conclusion: This cofactor specificity allows for rapid response to changing metabolic conditions. Significance: These findings help explain why mammals possess two distinct mitochondrial isozymes that switch expression during neural tube closure. Mammalian mitochondria are able to produce formate from cytoplasm, where the formate is reattached to tetrahydrofolate one-carbon donors such as serine, glycine, and sarcosine. This (THF) for use in de novo purine biosynthesis or further reduced pathway relies on the mitochondrial pool of tetrahydrofolate for either thymidylate synthesis or remethylation of homocys- (THF) and several folate-interconverting enzymes in the mito- teine to methionine. The 1C unit interconverting activities rep- chondrial matrix. We recently identified MTHFD2L as the resented in Fig. 1 by reactions 1–3 (1m–3m in mitochondria) enzyme that catalyzes the oxidation of 5,10-methylenetetrahy- are catalyzed by members of the methylenetetrahydrofolate drofolate (CH -THF) in adult mammalian mitochondria. We dehydrogenase (MTHFD) family in eukaryotes. The cytoplas- show here that the MTHFD2L enzyme is bifunctional, possess- mic MTHFD1 protein is a trifunctional enzyme possessing ing both CH -THF dehydrogenase and 5,10-methenyl-THF 10-formyl-THF (10-CHO-THF) synthetase, 5,10-methenyl- cyclohydrolase activities. The dehydrogenase activity can use THF (CH -THF) cyclohydrolase, and 5,10-methylene-THF either NAD or NADP but requires both phosphate and Mg (CH -THF) dehydrogenase activities (Fig. 1, reactions 1–3). when using NAD . The NADP -dependent dehydrogenase In contrast to the single trifunctional enzyme found in the activity is inhibited by inorganic phosphate. MTHFD2L uses the cytoplasm, three distinct MTHFD isozymes, encoded by three mono- and polyglutamylated forms of CH -THF with similar distinct genes, are now known to catalyze reactions 1m–3m catalytic efficiencies. Expression of the MTHFD2L transcript is (Fig. 1) in mammalian mitochondria. The final step in the mam- low in early mouse embryos but begins to increase at embryonic malian mitochondrial pathway to formate (Fig. 1, reaction 1m) day 10.5 and remains elevated through birth. In adults, is catalyzed by MTHFD1L, a monofunctional 10-CHO-THF MTHFD2L is expressed in all tissues examined, with the highest synthetase (2). The CH -THF dehydrogenase reaction (Fig. 1, levels observed in brain and lung. reaction 3m) is catalyzed by two homologous enzymes, MTHFD2 and MTHFD2L. MTHFD2 was initially identified in 1985 as an NAD -dependent 5,10-methylene-THF dehydro- Folate-dependent one-carbon (1C) metabolism is highly genase (3). Upon purification, this protein was found to be a bifunctional enzyme (4), also possessing CH -THF cyclohy- compartmentalized in eukaryotes, and mitochondria play a critical role in cellular 1C metabolism (reviewed in Ref. 1). The drolase activity (Fig. 1, reaction 2m). This enzyme, now referred cytoplasmic and mitochondrial compartments are metaboli- to as MTHFD2, has been extensively characterized with respect cally connected by the transport of 1C donors such as serine, to kinetics, substrate specificity, and expression profile (3, 5–10). glycine, and formate across the mitochondrial membranes in a mostly unidirectional flow (clockwise in Fig. 1). In mitochon- In 2011 we reported the discovery of a new mammalian mito- dria, the 1C units are oxidized to formate and released into the chondrial CH -THF dehydrogenase, termed MTHFD2L (11). MTHFD2L is homologous to MTHFD2, sharing 60–65% iden- tity. Recombinant rat MTHFD2L exhibits NADP -dependent * This work was supported, in whole or in part, by National Institutes of Health CH -THF dehydrogenase activity when expressed in yeast (11), Grants F32HD074428 (to J. M.) from the Eunice Kennedy Shriver NICHD and GM086856 (to D. R. A.) from NIGMS. but the enzyme was not purified in that study. In addition, it To whom correspondence should be addressed: Dept. of Molecular Biosci- could not be determined whether MTHFD2L was bifunctional ences, The University of Texas at Austin, Welch Hall A5300, Austin. TX because CH -THF cyclohydrolase assays are unreliable in 78712. Tel.: 512-471-5842; E-mail: [email protected]. The abbreviations used are: 1C, one carbon; THF, tetrahydrofolate; CH - crude extracts. This new isozyme is expressed in adult mito- THF, 5,10-methenyltetrahydrofolate; CH -THF, 5,10-methylenetetrahydro- chondria (11), whereas MTHFD2 is expressed only in trans- folate; 10-CHO-THF, 10-formyltetrahydrofolate; MTHFD, methylenetetra- formed mammalian cells and embryonic or nondifferentiated hydrofolate dehydrogenase; eEF2, eukaryotic translation elongation factor 2; TBP, TATA-box-binding protein. tissues (3). This is an open access article under the CC BY license. MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15507 MTHFD2L Exhibits Dual Redox Cofactor Specificity 5-CHO-THF (MP Biomedicals, Solon, OH) was conducted as described previously (16). Oligonucleotide primers were obtained from Integrated DNA Technologies (Coralville, IA). Tetrahydropteroylpentaglutamate (H PteGlu ) was prepared 4 5 by a modified NaBH reduction from the corresponding pteroylpentaglutamate (PteGlu ) (Schircks Laboratories, Jona, Switzerland), as described previously (17). Further preparation of 5,10-CH -H PteGlu was accomplished by incubation with 2 4 5 formaldehyde as described previously (14). Mice—All animals used within this study were maintained according to protocols approved by the Institutional Animal Care and Use Committee of The University of Texas at Austin and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Cloning of Rat MTHFD2L—RNA isolation and cDNA con- struction of rat MTHFD2L were conducted as described previ- FIGURE 1. Mammalian one-carbon metabolism. Reactions 1– 4 are in both ously (11). Removal of the mitochondrial targeting sequence the cytoplasmic and the mitochondrial (m) compartments. Reactions 1, 2, from the full-length rat MTHFD2L cDNA was accomplished by and 3, 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase, respectively, are catalyzed by trifunc- PCR amplification using primers rMTHFD2L1–40_forward tional C -THF synthase in the cytoplasm (MTHFD1). In mammalian mitochon- (5-GTCATCACCATCACCATCACGGATCCATGGCGAC- dria, reaction 1m is catalyzed by monofunctional MTHFD1L, and reactions 2m and 3m are catalyzed by bifunctional MTHFD2 or MTHFD2L. Reactions 4 GCGGGCC-3 and rMTHFD2L1–40_reverse (5-ACCTTA- and 4m are catalyzed by serine hydroxymethyltransferase and reaction 5 by GCGGCCGCAGATCTGGTACCCTAGTAGGTGATATTC- the glycine cleavage system. Gray ovals represent putative metabolite trans- T-3) (start codon is in bold, and underlined portions are porters. Hcy, homocysteine; dTMP, thymidylate. complementary to the rat MTHFD2L cDNA). The mitochond- The existence of these two CH -THF dehydrogenases rial targeting sequence (amino acids 1–40) was predicted by MitoProt (18). The resulting PCR-amplified fragment was (MTHFD2 and MTHFD2L) in mammalian mitochondria raises cloned into the pET22b vector (Novagen, EMD Biosciences). several questions. Do the two enzymes differ in their catalytic activity or in their substrate or cofactor specificity? Do they The pET22b-rMTHFD2L1–40 was further subcloned into a differ in their tissue distribution or expression profiles? To YEp24-based yeast expression vector containing the Saccha- romyces cerevisiae MET6 promoter using sequence- and ligat- answer these questions, we report here the purification and ion-independent cloning (17, 19). The YEp24 vector was mod- kinetic characterization of MTHFD2L and its embryonic and adult gene expression profiles. We show that MTHFD2L pos- ified to include a multiple cloning site and an N-terminal His sesses CH -THF cyclohydrolase activity and can use either tag (11). This construct (YEp24-rMTHFD2L1– 40) should produce a protein of 307 residues (N-terminal Met  2 Gly  6 NAD or NADP in its CH -THF dehydrogenase activity. His  298 MTHFD2L residues) with a calculated molecular MTHFD2L is expressed during mouse embryonic develop- ment, and there appears to be a switch from MTHFD2 to mass of 33,180 kDa. MTHFD2L expression at about the time of neural tube closure. Expression and Purification of MTHFD2L—The YEp24- rMTHFD2L1–40 construct was transformed into yeast strain A comparison of the enzymatic characteristics and expression MWY4.4 (ser1 ura3–52 trp1 his4 leu2 ade3–65mtd1) using a profiles of MTHFD2 and MTHFD2L revealed differences that may shed light on the roles of these two isozymes in adult and high efficiency lithium acetate yeast transformation procedure embryonic 1C metabolism. (20, 21). Transformed cells were grown in synthetic minimal medium (YMD) containing 0.7% (w/v) yeast nitrogen base EXPERIMENTAL PROCEDURES without amino acids (Difco Bacto) and 2% (w/v) glucose sup- Chemicals and Reagents—All reagents were of the highest plemented with L-serine (375 mg/liter), L-tryptophan (20 mg/li- commercial grade available. NAD and NADP were pur- ter), L-histidine (20 mg/liter), L-leucine (30 mg/liter), and ade- chased from U. S. Biological (Swampscott, MA) and Sigma, nine (20 mg/liter). Cultures were grown at 30 °C in a rotary respectively. HRP-conjugated goat anti-rabbit IgG was from shaker at 200 rpm and were harvested at 3–4 A by centrifu- Invitrogen, and the ECL Plus chemiluminescence detection kit gation at 8000  g for 5 min at 4 °C. The cell pellet was sus- was from GE Healthcare Life Sciences. Polyclonal antibodies pended in 2 ml of 25 mM Tris-Cl (pH 7.5) containing 1% (w/v) specific for MTHFD2L were produced in rabbits by the Dept. of sodium carbonate, 10 mM -mercaptoethanol, and 1 mM phe- Veterinary Sciences, M. D. Anderson Cancer Center, Bastrop, nylmethanesulfonyl fluoride (PMSF)/gram wet weight. The TX. THF was prepared by the hydrogenation of folic acid suspended cells were disrupted with glass beads using a Fast- (Sigma) using platinum oxide as a catalyst and purification of Prep FP120 cell disrupter (MP Biomedicals) followed by incu- the THF product on a DEAE cellulose column (Sigma) (12, 13). bation for 30 min at 4 °C to facilitate dissociation of MTHFD2L CH -THF was prepared nonenzymatically from THF and from cellular membranes. Cell debris was removed by centrif- formaldehyde (Fisher) (14). The yield of CH -THF was deter- ugation at 30,000  g for 30 min at 4 °C. The extracted mined by solving the equilibria of THF, formaldehyde, and MTHFD2L protein was then dialyzed at 4 °C overnight against -mercaptoethanol (15). The preparation of CH -THF from 25 mM Tris-Cl, 10 mM -mercaptoethanol, 1 mM PMSF, 20% 15508 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity TABLE 1 (v/v) glycerol, and 500 mM KCl at pH 8.5. Concentrated cell Primers used for gene expression profiling of the MTHFD gene family lysate (15 ml) was added to 9 ml of Ni -charged His-Bind in mouse embryos and adult organs resin (Novagen, Darmstadt, Germany) in 15 ml of binding Primer name Sequence Location buffer (final concentration: 25 mM Tris-Cl, 10 mM -mercapto- MTHFD1 F 5-TTCATCCCATGCACACCCAA-3 Exon 6 ethanol, 1 mM PMSF, 20% (v/v) glycerol, 500 mM KCl, and 20 MTHFD1 R 5-ATGCATGGGTGCACCAACTA-3 Exon 7 mM imidazole at pH 8.5). The slurry was mixed for3hat4 °C MTHFD1L F 5-GGACCCACTTTTGGAGTGAA-3 Exon 12 MTHFD1L R 5-ATGTCCCCAGTCAGGTGAAG-3 Exon 14 and then packed into a column. The column was washed at MTHFD2 F 5-ACAGATGGAGCTCACGAACG-3 Exon 5 room temperature with 40 column volumes of wash buffer con- MTHFD2 R 5-TGCCAGCGGCAGATATTACA-3 Exon 6 MTHFD2L F1 5-GGCGGGAAGATCCAAGAACG-3 Exon 6 taining 25 mM Tris-Cl, 10 mM -mercaptoethanol, 1 mM PMSF, MTHFD2L R1 5-CGCTATCGTCACCGTTGCAT-3 Exon 7 500 mM KCl, 10% (v/v) isopropanol, and 60 mM imidazole (pH MTHFD2L F2 5-GGCCAGCAGAGAGAAGAGACT-3 Exon 2 MTHFD2L R2 5-CCATGATTCCACTCCTTGCT-3 Exon 3 8.5) at a flow rate of 2–3 ml/min. His-tagged MTHFD2L was MTHFD2L F3 5-GAGGTGATGCAACGGTGAC-3 Exon 7 eluted at 4 °C at 0.5–1 ml/min with binding buffer containing MTHFD2L R3 5-GAATACCCGCAGCCACTATG-3 Exon 8 eEF2 F 5-CGCATCGTGGAGAACGTCAA-3 Exon 4 250 mM imidazole. Fractions containing active enzyme were eEF2 R 5-GCCAGAACCAAAGCCTACGG-3 Exon 5 determined by assaying for CH -THF dehydrogenase activity 2 TBP F 5-CATGGACCAGAACAACAGCC-3 Exon 2 TBP R 5-TAAGTCCTGTGCCGTAAGGC-3 Exon 3 (see below), and protein concentration was determined using the Bio-Rad protein assay reagent. Purified MTHFD2L was stored at 20 °C in 25 mM Tris-Cl, 10 mM -mercaptoethanol, tions at varying concentrations of NADP . Over the 5-min 100 mM KCl, and 50% (v/v) glycerol at a final pH of 7.5. Purity of period of measurement, 20% or less of the substrate was con- enzyme preparations was evaluated by SDS-PAGE. Enzyme verted to product, ensuring that initial rates were observed. K stability was confirmed by assaying CH -THF dehydrogenase was calculated from a global fit of the data to a competitive activity in aliquots of stored enzyme over a 1-week period. inhibition model using Prism. RNA Isolation and cDNA Synthesis—Tissue was collected The purified enzyme retained 75% 5,10-CH THF dehydro- genase activity after 1 week of storage, and all kinetic analyses from six male and six female C57BL/6 mice between 4 and 6 were performed within 1 week of enzyme purification. weeks of age. Embryos were dissected at the days indicated. For 5,10-Methylene-THF Dehydrogenase and 5,10-Methenyl- embryonic days 8.5–11.5, five embryos were pooled for each THF Cyclohydrolase Assays—A microplate assay was used for time point; for embryonic days 12.5–17.5, three embryos were determination of kinetic parameters as described previously pooled for each time point. Tissue and embryos were washed (22). CH -THF dehydrogenase activity was determined by an with PBS and then stored in RNAlater (Applied Biosystems Inc. end point assay (2). The reaction buffer consisted of 50 mM (ABI)). Embryos from each day were pooled prior to RNA iso- HEPES (pH 8.0), 100 mM KCl, 5 mM MgCl lation. RNA was isolated using TRI reagent (ABI) and treated , 0.4 mM CH -THF, 2 2 40 mM -mercaptoethanol, and either NAD (1 mM)or with TurboDnase (Invitrogen) following the manufacturer’s NADP (6 mM). Potassium phosphate (25 mM) was also instructions. RNA quality was verified by confirmation of the included for the NAD -dependent activity. To determine the presence of 28S and 18S rRNA bands on an agarose gel. cDNA was synthesized using SuperScript III (Invitrogen) and random cofactor specificity of the MTHFD2L enzyme in conditions that more closely resembled physiological conditions, reaction hexamers. buffer contained 50 mM HEPES (pH 8.0), 100 mM KCl, 0.5 mM Real-time PCR—Primers against mouse MTHFD1, MTHFD1L, MgCl ,10mM potassium phosphate, 40 mM -mercaptoetha- MTHFD2, MTHFD2L, eEF2 (eukaryotic translation elongation factor 2), and TBP (TATA-box-binding protein) were designed nol, and different cofactors including NAD (0.2 mM)or NADP (0.05 mM) and varying concentrations of CH -THF. using Primer-BLAST (25) to yield an amplicon between 95 and Sixty l of reaction mixture without CH -THF and 20 lof 125 base pairs long and cross at least one exon-exon junction purified MTHFD2L were mixed, and the enzyme reaction was (see Table 1). Primers were checked individually against plas- lofCH mids containing mouse MTHFD1, MTHFD1L, MTHFD2, and initiated by the addition of 20 -THF followed by incu- bation at 30 °C for 5 min. The reaction was quenched with 200 MTHFD2L to ensure that the primer pairs were specific to the l of 3% perchloric acid, and the plate was read at 350 nm on an target gene. Quantitative real-time PCR was performed using Infinite M200 (Tecan, Männedorf, Switzerland). The path SYBR Green (Qiagen) on an ABI ViiA 7 using a two-step pro- length was corrected using near-infrared measurements (23). gram (50 °C for 2 min and then 95 °C for 10 min followed by 40 CH -THF cyclohydrolase activity was determined by a con- cycles of 95 °C for 15 s and 60 °C for 15 s). The specificity of the tinuous assay (24) in microplate format. The enzyme reaction reaction was verified by melt curve analysis. Relative expression was incubated at 30 °C with MTHFD2L containing 200 mM values from embryos were calculated by normalizing to TBP potassium maleate (pH 7.4), 20 mM -mercaptoethanol, and (26), and values from adult tissues were normalized to eEF2 varying concentrations of CH -THF. The activity was moni- (27). tored by observing the decrease in absorbance of CH -THF at We have shown previously that MTHFD2L is alternatively 355 nm. spliced at exons 2 and 8 (11). To determine the abundance of the splice variants of MTHFD2L containing exon 2 or exon 8, For the determination of kinetic parameters, the initial rate data were fitted to the Michaelis-Menten equation by nonlinear we designed primer pairs that would bind in exons 2 and 3 or in regression using Prism (GraphPad, La Jolla, CA). Inhibition of exons 7 and 8, so that they would produce only a product from NADP -dependent dehydrogenase activity by phosphate ion splice variants containing exon 2 or exon 8, respectively. The expression values for these primer pairs were normalized to the was examined by assays at four fixed phosphate ion concentra- MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15509 MTHFD2L Exhibits Dual Redox Cofactor Specificity expression levels found using primers MTHFD2L F1 and R1, which bind in exons 6 and 7. There is no evidence for alternative splicing of these exons, and they are thus assumed to represent the total amount of MTHFD2L transcript, including all splice variants. In Situ Hybridization of Whole Mouse Embryos—Mouse embryos ranging from E9.5 to 12.5 were hybridized as described previously using digoxigenin-labeled UTP RNA probes (28). Antisense probes were constructed as described previously (28) by in vitro transcription using T7 RNA polymerase to tran- scribe from Riken clone 1110019K23 (29) linearized with BamHI. Sense probes were made by linearizing with XhoI and transcribing with T3 RNA polymerase. Yeast Complementation Assay—Exon 8 was removed from pET22b-rMTHFD2L by overlap extension PCR (30) using the primers rD2L-x8 5/HindIII F (5-ATCTAAGCTTATGGCG- ACGCGGGCCCGT-3), rD2L-x8 5 R(5-TAACAG CTGCA- GCCACTATGATAATCT-3), rD2L-x8 3 F(5-CTGCAGC- TGTTAAGAAGAAGGCCAGC-3), and rD2L-x8 3/BamHI R (V5) (5-TGAT GGATCC A GTAGGTGATATTCTTGGCA- GCCAG-3). Following PCR, rMTHFD2L-x8 was subcloned into YEp24ES (11, 17) using the HindIII and BamHI restriction sites. Plasmids were transformed into the yeast strain MWY4.5 (ser1 ura3–52 trp1 leu2 his4 ade3–30/65 mtd1), and transformants were selected for uracil prototrophy. The in vivo complementation assay was performed in this yeast strain as described previously (11). Preparation of crude yeast lysates and immunoblotting were carried out as described previously (11). The protein concen- tration was determined by BCA assay (Thermo Fisher Scien- tific). NAD -dependent CH -THF dehydrogenase activity in the lysates was assayed with MgCl and potassium phosphate as described above. RESULTS Expression and Purification of Rat MTHFD2L—Our initial characterization of mammalian MTHFD2L relied on the recombinant full-length rat protein expressed in yeast crude extracts. Difficulties in purification, likely because of its tight association with membranes (11), prevented us from conduct- ing a full enzymatic characterization of MTHFD2L in that FIGURE 2. Purified rat MTHFD2L possesses cyclohydrolase activity. A, study. In the present study, we overcame these problems by His -tagged rat MTHFD2L was purified by immobilized nickel affinity chroma- tography as described under “Experimental Procedures.” Lane 1 shows the using several strategies. To increase the yield of recombinant imidazole elution after washing the column at 4 °C. In a second experiment, protein, we replaced the mitochondrial targeting sequence of the column was washed at room temperature instead of 4 °C. Lane 2 shows the room temperature wash fractions. Lane 3 shows the imidazole elution the rat MTHFD2L with an N-terminal His tag for localization after washing at room temperature. B, dependence of cyclohydrolase activity in the yeast cytosol (31). Despite the loss of its targeting on (6R,S)5,10-methenyl-THF concentration. Results shown are the means sequence, the protein remained associated with cellular mem- S.E. of five replicates (error bars are included for all data points but are obscured by the data symbol when the scatter is small). branes; so we included sodium carbonate in the extraction buffer to release MTHFD2L from membranes (32). Finally, we washed the Ni column at room temperature rather than at The specific activity shown in Fig. 2B is 11-fold higher than 4 °C to eliminate contamination from endogenous yeast pro- the buffer-catalyzed rate. Cyclohydrolase activity could not be teins (33). This washing step allowed the purification of determined at saturating conditions due to the high absorbance MTHFD2L to greater than 95% homogeneity (Fig. 2A, cf. lanes of substrate, and thus accurate values for k and K could not cat m 1 and 3). The mobility of the purified protein is consistent with be calculated. the expected size of 33 KDa. Steady-state Kinetics and Cofactor Specificity of 5,10-Methyl- MTHFD2L Possesses 5,10-Methenyl-THF Cyclohydrolase ene-THF Dehydrogenase Activity—We reported previously that Activity—MTHFD2L exhibits robust CH -THF cyclohydro- recombinant full-length rat MTHFD2L expressed in yeast exhibits NADP -dependent CH -THF dehydrogenase activity lase activity, confirming the bifunctional nature of this enzyme. 15510 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity TABLE 2 Kinetic parameters for MTHFD2L 5,10-methylene-THF dehydrogen- ase activity Enzyme assays were performed as described under “Experimental Procedures.” CH -THF kinetic parameters were determined using saturating concentrations of NAD (1.0 mM) or NADP (6.0 mM). When NAD was used, potassium phosphate (25 mM) and MgCl (5 mM) were also included. Redox cofactor kinetic parameters were determined using saturating concentrations of CH -H PteGlu (400 M). 2 4 1 Substrate NAD /NADP CH THF k k /K 2 cat cat m 1 1 1 M s s M NAD 147  16 3.6  0.1 0.025 CH -H PteGlu 40  5 2.7  0.07 0.067 2 4 1 CH -H PteGlu 130  30 8.8  0.4 0.068 2 4 5 NADP 537  54 1.1  0.04 0.002 CH -H PteGlu 42  7 1.3  0.05 0.030 2 4 1 CH -H PteGlu 153  39 7.2  0.5 0.047 2 4 5 in crude extracts (11). Purified MTHFD2L exhibited both NAD - and NADP -dependent CH -THF dehydrogenase activity using either mono- or pentaglutamylated CH -THF (CH -H PteGlu and CH -H PteGlu , respectively). The 2 4 1 2 4 5 kinetic parameters are given in Table 2. Values for k and K cat m differed between the polyglutamylation states of the CH -THF substrate, but they were not sensitive to the redox cofactor in the dehydrogenase reaction (NAD or NADP ). The K values for CH -H PteGlu were 3.5 times higher than those of CH - 2 4 5 2 H PteGlu regardless of the redox cofactor. However, this was 4 1 accompanied by a similar increase in k , resulting in similar cat k /K values (Table 2). Using CH -H PteGlu , the K values cat m 2 4 1 m for NAD and NADP were 147  16 and 537  54 M, respectively (Table 2). Although the dehydrogenase activity of the bifunctional MTHFD2 (an isozyme of MTHFD2L) is classified as NAD - FIGURE 3. 5,10-Methylene-THF dehydrogenase activity of MTHFD2L. A, specific, it can also utilize NADP at a reduced efficiency (6). purified MTHFD2L was assayed for NAD - and NADP -dependent 5,10-methyl- The NAD -dependent activity of MTHFD2 requires Mg and 2 ene-THF dehydrogenase activity in the presence or absence of P and Mg . Enzyme activity is expressed as mol product/min/mg of protein. Each column inorganic phosphate (P ) (6). To explore redox cofactor speci- represents the mean  S.E. of triplicate determinations. Reaction buffer con- ficity in MTHFD2L, we measured specific activity using satu- tained 50 mM HEPES (pH 8.0), 100 mM KCl, 0.4 mM 5,10-CH -THF, and 40 mM rating levels of NAD or NADP in the presence of Mg -mercaptoethanol. NAD was used at 1.0 mM and NADP at 6.0 mM.Mg and were tested by including 5.0 mM MgCl and 25 mM potassium phosphate and/or P (Fig. 3A). When NAD was used, the dehydrogenase i 2 where indicated. B, inhibition of NADP -dependent 5,10-methylene-THF dehy- activity of MTHFD2L was strongly dependent on the presence drogenase activity by inorganic phosphate. Each point represents the mean of Mg and P in combination. Using CH -H PteGlu and S.E. of triplicate determinations (error bars are included for all data points but are i 2 4 1 obscured by the data symbol when the scatter is small). The curves represent NAD , the K values for Mg and P were 233 62 and 293 m i nonlinear fits to the Michaelis-Menten model. The reaction buffer (as in A) con- 59 M, respectively. When NADP was used, MTHFD2L activ- tained 5.0 mM MgCl and varying concentrations of NADP . Potassium phos- phate concentrations were 0 (●),5(), 10 (Œ), and 25 (ƒ)mM. ity was increased slightly with Mg . Inorganic phosphate appeared to counteract the Mg effect with NADP (Fig. 3A). Because MTHFD2 shows inhibition of NADP -dependent see the legend to Fig. 4 for references). For each component, the dehydrogenase activity by inorganic phosphate (6), we sought ratio of NAD -dependent to NADP -dependent dehydrogen- to determine if MTHFD2L behaves in the same way. We ase activities was plotted (Fig. 4). The data indicate that there is observed that increasing concentrations of phosphate ion an 3.5-fold preference for NAD at physiological P concen- reduced the NADP -dependent dehydrogenase activity of trations (Fig. 4A) and a 5–6-fold preference for NAD at phys- MTHFD2L (Fig. 3B). The data fit best to a competitive inhibi- iological Mg concentrations (Fig. 4B). At high concentra- tion model with a K of 1.9  0.3 mM. tions of CH -THF, MTHFD2L clearly prefers NAD (Fig. 4C). i 2 Cofactor Specificity at Physiological Concentrations of Mg , However, as the CH -THF concentration is lowered to more P , and CH -THF—To better understand the cofactor prefer- physiological levels, the ratio approaches 1, and at the lowest i 2 ence (NAD versus NADP ) of the MTHFD2L dehydrogenase CH -THF concentrations, the enzyme is more active with activity under physiologically relevant substrate conditions, we NADP . This suggests that cofactor preference will be very repeated the assay at a wide range of P ,Mg , and CH -THF sensitive to CH -THF concentration in vivo. i 2 2 concentrations. The range used includes concentrations esti- Gene Expression Profile of MTHFD Gene Family in Mouse mated to exist in the matrix compartment of mammalian mito- Embryos—It has been reported previously that the MTHFD2 chondria (CH gene is expressed in early embryos, but its expression declines THF 2.5–25 M,Mg 0.5 mM,P 10 mM; 2- i MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15511 MTHFD2L Exhibits Dual Redox Cofactor Specificity FIGURE 5. Temporal expression profile of MTHFD gene family in mouse embryos. The relative expression profiles of MTHFD1 (●), MTHFD1L (f), MTHFD2 (Œ), and MTHFD2L () was determined by real-time PCR as described under “Experimental Procedures.” The age of the embryos from which the RNA was obtained is indicated in embryonic days (birth occurs at E20.0). mRNA expression was normalized to that of the TBP transcript. Each point represents the mean  S.E. of triplicate determinations (error bars are included for all data points but are obscured by the data symbol when the scatter is small). as the embryo approaches birth, and the MTHFD2 enzyme is found only in nondifferentiated tissues in adults (9, 28). We determined the relative expression profiles of all four MTHFD gene family members in mouse embryos using real-time PCR, normalizing to TBP (TATA-box-binding protein) expression. TBP has been determined to be stably expressed at multiple stages of embryonic development, making it a suitable house- keeping gene for real-time PCR experiments involving embryos (26). MTHFD1 and MTHFD1L transcript expression was high- est at E8.5 and subsequently decreased until expression increased again at E13.5–17.5 (Fig. 5). MTHFD2 expression was low at all embryonic days examined. MTHFD2L was also expressed at all embryonic days examined. MTHFD2L expres- sion was low at E8.5 but increased beginning at E10.5 (Fig. 5). These profiles suggest that a switch between MTHFD2 and MTHFD2L expression occurs approximately between embry- onic days 8.5 and 10.5 during mouse embryogenesis. In situ hybridization of whole mouse embryos (Fig. 6) revealed that MTHFD2L is expressed in the neural tube and the forebrain, midbrain, and hindbrain, suggesting a possible role in neural tube development. Other areas of intense staining included the branchial arches and limb buds, particularly along the progress zone. Gene Expression Profile of MTHFD Gene Family in Adult Mice—Expression of adult MTHFD genes from male and female mice was normalized to the expression of eEF2, a house- keeping gene that is stably expressed in a wide array of adult tissues (27). The MTHFD2L transcript was expressed in all of the tissues examined, with the highest expression observed in FIGURE 4. Redox cofactor specificity of MTHFD2L at physiological con- centrations of P ,Mg , and 5,10-CH -THF. The ratio of NAD - to NADP - i 2 dependent 5,10-methylene-THF activity was plotted as a function of increas- dent reactions. For the NADP -dependent reactions, only 5 mM MgCl was ing concentrations of inorganic phosphate (A), Mg (B), or 5,10-CH -THF (C). included. Reported mitochondrial matrix substrate concentration ranges Reaction buffer (described in the legend for Fig. 3) contained either 1 mM (indicated by shaded areas) are 5–15 mM for P (47– 49), 0.3– 0.4 mM for Mg NAD or6mM NADP . 5,10-CH -THF was included at 0.4 mM (A and B)or (48, 49), and 2.5–25 M for 5,10-CH THF (37– 40), respectively. The best esti- 2 2 varied (C). For dependence on P mates found for in vivo mitochondrial NADP concentration (A),5mM MgCl was included and NAD concentrations are i 2 for both NAD - and NADP -dependent reactions. For dependence on Mg NADP 80 M and NAD 240 M (42, 43). The data in C were fit to the concentration (B), 25 mM potassium phosphate was included in NAD Michaelis-Menten equation. The 5,10-CH -de- -THF concentration that gave the pendent but not NADP -dependent reactions. For dependence on 5,10-CH - half-maximal ratio of NAD - to NADP -dependent 5,10-methylene-THF THF concentration, 5 mM MgCl activity was 12.5 M. and 25 mM P were included in NAD -depen- 2 i 15512 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity FIGURE 6. Spatial distribution of MTHFD2L expression in mouse embryos. In situ hybridizations of whole mount embryos ranging from E9.5 to E12.5 (A–D) were performed using digoxigenin-labeled UTP RNA probes as described under “Experimental Procedures.” C, as shown on the E11.5 embryo, MTHFD2L is especially prominent in the neural tube (a), forebrain (b), midbrain (c), hindbrain (d), branchial arches (e), and somites (f). Embryos A–C were imaged at the same magnification (20). Embryo D was imaged at 12.5 magnification. Scale bars correspond to 1 mm. FIGURE 8. MTFHD2L splice variants during mouse development. The rel- ative abundance of exon 2- (f) and exon 8-containing (●) MTHFD2L tran- scripts was determined by real-time PCR and analyzed as described under “Experimental Procedures.” Each point represents the mean  S.E. of tripli- cate determinations (‘bars are included for all data points but are obscured by the data symbol when the scatter is small). Alternative Splicing of MTHFD2L—We have shown previ- ously that MTHFD2L is alternatively spliced at exons 2 and 8 (11), with either exon skipped or included in the mature mRNA. The skipping of exon 2 and inclusion of exon 8 produces an enzymatically active enzyme of 347 residues (including the 40-residue mitochondrial targeting sequence) (11). The inclu- sion of exon 2 results in the introduction of an early stop codon. However, translation beginning from an ATG in exon 3 is pre- dicted to produce a truncated protein missing the mitochon- drial targeting sequence. When exon 8 is skipped, exon 7 is spliced to exon 9 with the reading frame intact, resulting in FIGURE 7. Expression profile of MTHFD gene family in adult mouse tis- deletion of 43 codons. Using total embryo RNA from embryos sues. The relative expression profiles of MTHFD1 (green), MTHFD1L (red), ages E8.5 to E17.5, we observed that exon 2 was present in less MTHFD2 (blue), and MTHFD2L (black) was determined by real-time PCR as than 10% of the transcripts in all of the embryonic days exam- described under “Experimental Procedures.” mRNA expression was normal- ized to that of the eEF2 transcript in female (top) and male (bottom) adult ined (Fig. 8), indicating that the vast majority of MTHFD2L mice. Each column represents the mean  S.E. of triplicate determinations. transcripts encode a mitochondrial targeting sequence. Exon 8, brain and lung and lower expression in liver and kidney (Fig. 7). on the other hand, was found to be missing in 20–45% of the As has been reported previously, MTHFD1 showed the highest MTHFD2L transcripts throughout embryogenesis (Fig. 8). expression in liver and kidney (34). MTHFD1L was most highly To determine what effect the removal of exon 8 would have expressed in brain and spleen. The MTHFD2 transcript could on MTHFD2L activity, MTHFD2L lacking exon 8 was be detected at low levels in most tissues, but only testis and expressed in yeast. NAD -dependent CH -THF dehydroge- spleen expressed it at even a modest level. The expression pat- nase activity could not be detected in crude yeast lysate from terns of the four transcripts were similar in males and females. yeast cells transformed with the truncated construct (YEp- MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15513 MTHFD2L Exhibits Dual Redox Cofactor Specificity sesses both CH -THF dehydrogenase and CH -THF cyclohy- drolase activities (Fig. 2). The dehydrogenase activity of this bifunctional enzyme can use either NAD or NADP but requires both phosphate and Mg when using NAD (Fig. 3A). The NADP -dependent dehydrogenase activity of MTHFD2L is inhibited by inorganic phosphate (Fig. 3B). MTHFD2L can use the mono- and polyglutamylated forms of CH -THF with similar catalytic efficiencies (k /K ; Table 2). 2 cat m Expression of the MTHFD2L transcript is low in early mouse embryos, begins to increase at E10.5, and continues through birth (Fig. 5). In adults, MTHFD2L was expressed in all tissues examined, with the highest levels observed in brain and lung (Fig. 7). How do these cofactor requirements compare with those of the other CH -THF dehydrogenase/CH -THF cyclohydrolase found in mammalian mitochondria? The MTFHD2 isozyme FIGURE 9. Expression of MTHFD2L lacking exon 8 in yeast. S. cerevisiae strain has been named NAD -dependent methylenetetrahydrofolate MWY4.5 (ser1 ura3 trp1 leu2 his4 ade3–30/65 mtd1) was transformed to uracil dehydrogenase-methenyltetrahydrofolate cyclohydrolase (6, prototrophy with YEp-rD2L (wild type), YEp-rD2L-x8 (lacking exon 8), or empty 10) but in fact exhibits dehydrogenase activity with NADP , vector (YEp24ES). Ura transformants were streaked onto yeast minimal plates containing serine as a one-carbon donor plus adenine (left) or serine alone (right) albeit with a much higher K and lower V (6). In fact, the m max and incubated at 30 °C for 4 days. Both plates also contained leucine, tryptophan, redox cofactor requirements of the two isozymes are quite sim- and histidine to support the other auxotrophic requirements of MWY4.5. Inset, ilar: both exhibit lower K values for NAD than for NADP ; immunoblot of whole cell lysate from MWY4.5 transformed with the indicated plasmids. Each lane was loaded with 50 g of protein. The blot was probed with their NAD -dependent activities require phosphate and Mg ; polyclonal antibodies against MTHFD2L (1:1000 dilution). and their NADP -dependent activities are inhibited by phos- phate. The absolute requirement of the NAD -dependent rD2L-x8; data not shown). NAD -dependent CH -THF dehy- activity of MTHFD2 for Mg and P has been characterized in 2 i great detail by Mackenzie and co-workers (10). MTHFD2 uses drogenase activity was easily detectable in lysate from yeast cells transformed with the full-length construct. Mg and P to convert an NADP binding site into an NAD We next asked whether MTHFD2L lacking exon 8 was active binding site. P binds in close proximity to the 2-hydroxyl of in vivo, using a yeast complementation assay (11). Briefly, this NAD and competes with NADP binding. Mg plays a role in positioning P and NAD. Mackenzie and co-workers (10) iden- assay uses yeast strain MWY4.5, which lacks cytoplasmic CH - THF dehydrogenase activities as well as the 10-formyl-THF tified several amino acid residues in MTHFD2 that are involved synthetase activity of the cytoplasmic trifunctional C -THF in the P and Mg binding, and these residues are highly con- 1 i synthase (35). Wild-type yeast can produce 10-CHO-THF for served in MTHFD2L in mammals. It is thus likely that Mg and P play a mechanistically similar role in the NAD -depen- de novo purine biosynthesis from serine using either cytoplas- mic or mitochondrial 1C pathways (36) (see Fig. 1). However, dent dehydrogenase activity of MTHFD2L as well. MWY4.5 is blocked in both pathways to 10-CHO-THF. This At saturating levels of CH -THF, using NAD as a cofactor, blockage creates a requirement for adenine in the growth the k /K value for the CH -THF dehydrogenase reaction of cat m 2 1 1 MTHFD2 is 2.9 s M (8). This value is 45-fold higher medium (35). Cytoplasmically localized MTHFD2L lacking exon 8 should rescue the adenine requirement of MWY4.5 if it is cata- than the efficiency exhibited by MTHFD2L (k /K 0.067 cat m 1 1 lytically active in vivo. Transformants of MWY4.5 harboring YEp- s M ). These differences in kinetic parameters are unlikely rD2L-x8, YEp-rD2L (full-length MTHFD2L) (11), or empty vector to be due to different assay conditions. When we performed the CH -THF dehydrogenase assay under buffer conditions that (YEp24ES) were streaked onto yeast minimal plates containing serine as a one-carbon donor or serine adenine and incubated at were used to characterize MTHFD2 (MOPS (pH 7.3)) rather 30 °C. As shown in Fig. 9, full-length MTHFD2L, expressed from than the HEPES (pH 8.0) used in this article, we did not observe significant changes in the values for k YEp-rD2L as a positive control, fully complemented the adenine or K (data not shown). cat m The apparent preference of MTHFD2L for NAD (Fig. 3A)is requirement of MWY4.5, as observed previously (11). However, MTHFD2L lacking exon 8, expressed from the rD2L-x8 plasmid, most dramatic at nonphysiological levels of phosphate, Mg , did not complement the adenine requirement. An immunoblot of and folate cofactor. When these experiments were repeated crude yeast lysates from these transformants confirmed that the at more physiologically relevant substrate concentrations, MTHFD2L showed much less preference for NAD (Fig. 4). In truncated protein was expressed at a level similar to that of the full-length protein in control cells (Fig. 9, inset). These in vitro and fact, at CH -THF concentrations below 10 M, the enzyme is in vivo results suggest that the MTHFD2L variant lacking exon 8 more active with NADP than with NAD (Fig. 4C). Given that does not function as a CH -THF dehydrogenase. estimates for mitochondrial matrix levels of CH -THF range 2 2 from 2.5 to 25 M (37–40), it is likely that MTHFD2L exhibits DISUSSION dual redox cofactor specificity in vivo. The experiments described here demonstrate that the mam- The use of NAD versus NADP in this step can have a malian MTHFD2L isozyme, like MTHFD1 and MTHFD2, pos- dramatic effect on the rate and direction of flux of one-carbon 15514 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity units through this pathway in mitochondria. The oxidation tion for gluconeogenesis. When CH -THF dehydrogenase and state of mitochondrial pools of NAD and NADP is dictated CH -THF cyclohydrolase activities are included in the model, by mitochondrial respiration (41), which in turn is linked to the mitochondrial folate pathway produces formate for cyto- nutrition, differentiation, and proliferation (42). Measurements solic export, where it is incorporated into purines, thymidylate, in liver suggest that the redox potential of the NAD /NADH and the methyl cycle (37). Christensen and MacKenzie (44) have proposed that the level of MTHFD2 expression could act matrix pool is typically 75–100 mV more positive than that of the NADP /NADPH matrix pool (41, 43). Thus, the ratio of as a metabolic switch to control the balance between serine and CH -THF to 10-CHO-THF in the matrix (reactions 3m and 2m formate production. in Fig. 1) will be shifted much further toward 10-CHO-THF We suggest that the existence of two mitochondrial dehydro- genase/cyclohydrolase isozymes in mammals (MTHFD2 and with a CH -THF dehydrogenase linked to the NAD /NADH pool versus the NADP /NADPH pool (6, 7). A cyclohydrolase/ MTHFD2L) reflects the need to tightly regulate flux through dehydrogenase with dual cofactor specificity, such as this oxidation step in response to changing metabolic condi- MTHFD2L, would be able to adapt immediately to changing tions and needs. For example, de novo purine biosynthesis is metabolic conditions, shifting the equilibrium between CH especially important in rapidly dividing cells, such as during THF and 10-CHO-THF (and formate) depending on the rela- embryogenesis. Thus, early embryos express both MTHFD2 tive levels of oxidized cofactor (NAD or NADP ) in the mito- and MTHFD2L isozymes, ensuring that mitochondrial formate chondrial matrix. We do not know whether the MTHFD2 production is adequate to support de novo purine biosynthesis. Indeed, embryonic growth and neural tube closure requires isozyme might also exhibit dual redox cofactor specificity in vivo, as it was not characterized at physiologically relevant sub- mitochondrial formate production (45). Compared with strate concentrations (6, 10). embryos, however, adult mammals do not have a high demand We determined expression profiles for the entire MTHFD for de novo purine biosynthesis (46). The loss of expression of family of genes during mouse embryogenesis (Fig. 5) and in MTHFD2 as the embryos approach birth may reflect the lower adult tissues (Fig. 7). The results for MTHFD1 (cytoplasmic demand for de novo purine biosynthesis in neonate and adult reactions 1–3 (Fig. 1)) and MTHFD1L (mitochondrial reaction mammals. 1m) are qualitatively similar to previously reported transcript In addition to switching between expressing one or two expression patterns in mouse embryos based on a staged mitochondrial CH -THF dehydrogenase/CH -THF cyclohy- Northern blot (28). Both transcripts are highest in early drolase enzymes, there may be other ways of regulating embryos and decrease during embryonic days 9.5–15.5, only to MTHFD2L expression such as alternative splicing. We have increase again as the embryo approaches birth. MTHFD2 and shown previously that an exon 8 deletion is present in adult MTHFD2L, on the other hand, exhibit very different expression tissues (11). We observed significant expression of this same profiles. MTHFD2 expression was low in all embryonic days splice variant of MTHFD2L throughout embryogenesis (Fig. 8). examined, whereas expression of the MTHFD2L transcript However the protein that would be produced from the tran- increased beginning at E10.5 and remained elevated through script lacking exon 8 did not show activity in vitro or in vivo (Fig. birth (Fig. 5). These data reveal a switch from MTHFD2 to 9), and the function of this splice variant is unknown. MTHFD2L expression at about the time of neural tube closure in mouse embryos. The spatial expression of MTHFD2L is Acknowledgments—We thank Nafee Talukder for assistance with the localized to the neural tube, developing brain, branchial arches, purification of recombinant MTHFD2L and Jordan Lewandowski for and limb buds (Fig. 6). These regions are also areas where performing the in situ hybridizations. MTHFD2 and MTHFD1L are expressed (28), suggesting a role for the mitochondrial folate pathway in these embryonic REFERENCES tissues. 1. Tibbetts, A. S., and Appling, D. R. (2010) Compartmentalization of mam- Why might mammals possess these two distinct mitochon- malian folate-mediated one-carbon metabolism. Annu. Rev. Nutr. 30, drial dehydrogenase/cyclohydrolase isozymes? It appears that 57–81 under most conditions, the majority of 1C units for cytoplasmic 2. Walkup, A. S., and Appling, D. R. (2005) Enzymatic characterization of processes are derived from mitochondrial formate (1). Oxida- human mitochondrial C1-tetrahydrofolate synthase. Arch. Biochem. Bio- tion of mitochondrial CH -THF is essential for the production phys. 442, 196–205 3. Mejia, N. R., and MacKenzie, R. E. (1985) NAD-dependent methylenetet- of 10-CHO-THF, which is processed by MTHFD1L to provide rahydrofolate dehydrogenase is expressed by immortal cells. J. Biol. Chem. formate for cytoplasmic export (Fig. 1). This formate is then 260, 14616–14620 reattached to THF for use in de novo purine biosynthesis or 4. Mejia, N. R., Rios-Orlandi, E. M., and MacKenzie, R. E. (1986) NAD-de- further reduced for either thymidylate synthesis or remethyla- pendent methylenetetrahydrofolate dehydrogenase-methenyltetrahydro- tion of homocysteine to methionine. Modeling studies suggest folate cyclohydrolase from ascites tumor cells: purification and properties. that the oxidation step (reaction 3m (Fig. 1)), catalyzed by J. Biol. Chem. 261, 9509–9513 5. Mejia, N. R., and MacKenzie, R. E. (1988) NAD-dependent methylenetet- either MTHFD2 or MTHFD2L, is a critical control point for rahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase mitochondrial 1C metabolism. Using an in silico model, in transformed cells is a mitochondrial enzyme. Biochem. Biophys. Res. Nijhout et al. (37) observed that the exclusion of CH -THF Comm. 155, 1–6 dehydrogenase and CH -THF cyclohydrolase activities from 6. Yang, X. M., and MacKenzie, R. E. (1993) NAD-dependent methylenetet- the mitochondrial folate pathway results in loss of formate rahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the export and a dramatic increase in mitochondrial serine produc- MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15515 MTHFD2L Exhibits Dual Redox Cofactor Specificity yeast MIS1 gene. Biochemistry 32, 11118–11123 28. Pike, S. T., Rajendra, R., Artzt, K., and Appling, D. R. (2010) Mitochondrial 7. Pelletier, J. N., and MacKenzie, R. E. (1995) Binding and interconversion of C1-THF synthase (MTHFD1L) supports flow of mitochondrial one-car- tetrahydrofolates at a single site in the bifunctional methylenetetrahydro- bon units into the methyl cycle in embryos. J. Biol. Chem. 285, 4612–4620 folate dehydrogenase/cyclohydrolase. Biochemistry 34, 12673–12680 29. Kawai, J., Shinagawa, A., Shibata, K., Yoshinom M., Itoh, M., Ishii, Y., 8. Pawelek, P. D., and MacKenzie, R. E. (1998) Methenyltetrahydrofolate Arakawa, T., Hara, A., Fukunishi, Y., Konno, H., Adachi, J., Fukuda, S., cyclohydrolase is rate limiting for the enzymatic conversion of 10-form- Aizawa, K., Izawa, M., Nishi, K., Kiyosawa, H., Kondo, S., Yamanaka, I., yltetrahydrofolate to 5,10- methylenetetrahydrofolate in bifunctional de- Saito, T., Okazaki, Y., Gojobori, T., Bono, H., Kasukawa, T., Saito, R., hydrogenase-cyclohydrolase enzymes. Biochemistry 37, 1109–1115 Kadota, K., Matsuda, H., Ashburner, M., Batalov, S., Casavant, T., Fleis- 9. Di Pietro, E., Wang, X. L., and MacKenzie, R. E. (2004) The expression of chmann, W., Gaasterland, T., Gissi, C., King, B., Kochiwa, H., Kuehl, P., mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase Lewis, S., Matsuo, Y., Nikaido, I., Pesole, G., Quackenbush, J., Schriml, supports a role in rapid cell growth. Biochim. Biophys. Acta 1674, 78–84 L. M., Staubli, F., Suzuki, R., Tomita, M., Wagner, L., Washio, T., Sakai, K., 10. Christensen, K. E., Mirza, I. A., Berghuis, A. M., and Mackenzie, R. E. Okido, T., Furuno, M., Aono, H., Baldarelli, R., Barsh, G., Blake, J., Boffelli, (2005) Magnesium and phosphate ions enable NAD binding to methyl- D., Bojunga, N., Carninci, P., de Bonaldo, M. F., Brownstein, M. J., Bult, C., enetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohy- Fletcher, C., Fujita, M., Gariboldi, M., Gustincich, S., Hill, D., Hofmann, drolase. J. Biol. Chem. 280, 34316–34323 M., Hume, D. A., Kamiya, M., Lee, N. H., Lyons, P., Marchionni, L., 11. Bolusani, S., Young, B. A., Cole, N. A., Tibbetts, A. S., Momb, J., Bryant, Mashima, J., Mazzarelli, J., Mombaerts, P., Nordone, P., Ring, B., Ringwald, J. D., Solmonson, A., and Appling, D. R. (2011) Mammalian MTHFD2L M., Rodriguez, I., Sakamoto, N., Sasaki, H., Sato, K., Schönbach, C., Seya, Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase T., Shibata, Y., Storch, K. F., Suzuki, H., Toyo-oka, K., Wang, K. H., Weitz, isozyme expressed in adult tissues. J. Biol. Chem. 286, 5166–5174 C., Whittaker, C., Wilming, L., Wynshaw-Boris, A., Yoshida, K., Hase- 12. Blakley, R. L. (1957) The interconversion of serine and glycine: prepara- gawa, Y., Kawaji, H., Kohtsuki, S., Hayashizaki, Y, RIKEN Genome Explo- tion and properties of catalytic derivatives of pteroylglutamic acid. ration Research Group Phase II Team, and the FANTOM Consortium Biochem. J. 65, 331–342 (2001) Functional annotation of a full-length mouse cDNA collection. 13. Curthoys, N. P., and Rabinowitz, J. C. (1971) Formyltetrahydrofolate syn- Nature 409, 685–690 thetase: binding of adenosine triphosphate and related ligands determined 30. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J.K., and Pease, L. R. (1989) by partition equilibrium. J. Biol. Chem. 246, 6942–6952 Site-directed mutagenesis by overlap extension using the polymerase 14. Appling, D. R., and West, M. G. (1997) Monofunctional NAD-dependent, chain reaction. Gene. 77, 51–59 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces 31. Zhang, L., Joshi, A. K., and Smith, S. (2003) Cloning, expression, charac- cerevisiae. Methods Enzymol. 281, 178–188 terization, and interaction of two components of a human mitochondrial 15. Kallen, R. G., and Jencks, W. P. (1966) The mechanism of the condensa- fatty acid synthase: malonyltransferase and acyl carrier protein. J. Biol. tion of formaldehyde with tetrahydrofolic acid. J. Biol. Chem. 241, Chem. 278, 40067–40074 5851–5863 32. Prasannan, P., and Appling, D. R. (2009) Human mitochondrial C1-tetra- 16. Rabinowitz, J. C. (1963) Preparation and properties of 5,10-methenyltet- hydrofolate synthase: submitochondrial localization of the full-length en- rahydrofolic acid and 10-formyltetrahydrofolic acid. Methods Enzymol. 6, zyme and characterization of a short isoform. Arch. Biochem. Biophys. 814–815 481, 86–93 17. Suliman, H. S., Sawyer, G. M., Appling, D. R., and Robertus, J. D. (2005) 33. Dihazi, H., Kessler, R., and Eschrich, K. (2001) One-step purification of Purification and properties of cobalamin-independent methionine syn- recombinant yeast 6-phosphofructo-2-kinase after the identification of thase from Candida albicans and Saccharomyces cerevisiae. Arch. contaminants by MALDI-TOF MS. Protein Expr. Purif. 21, 201–209 Biochem. Biophys. 441, 56–63 34. Thigpen, A. E., West, M. G., and Appling, D. R. (1990) Rat C -tetrahydro- 18. Claros, M. G., and Vincens, P. (1996) Computational method to predict folate synthase: cDNA isolation, tissue-specific levels of the mRNA, and mitochondrially imported proteins and their targeting sequences. Eur. expression of the protein in yeast. J. Biol. Chem. 265, 7907–7913 J. Biochem. 241, 779–786 35. West, M. G., Horne, D. W., and Appling, D. R. (1996) Metabolic role of 19. Li, M. Z., and Elledge, S. J. (2007) Harnessing homologous recombination cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in vitro to generate recombinant DNA via SLIC. Nat. Methods 4, 251–256 in Saccharomyces cerevisiae. Biochemistry 35, 3122–3132 20. West, M. G., Barlowe, C. K., and Appling, D. R. (1993) Cloning and char- 36. Barlowe, C. K., and Appling, D. R. (1990) Molecular genetic analysis of acterization of the Saccharomyces cerevisiae gene encoding NAD-depen- Saccharomyces cerevisiae C -tetrahydrofolate synthase mutants reveals a dent 5,10-methylenetetrahydrofolate dehydrogenase. J. Biol. Chem. 268, noncatalytic function of the ADE3 gene product and an additional folate- 153–160 dependent enzyme. Mol. Cell. Biol. 10, 5679–5687 21. Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium 37. Nijhout, H. F., Reed, M. C., Lam, S. L., Shane, B., Gregory, J. F., 3rd, and acetate/single-stranded carrier DNA/polyethylene glycol method. Meth- Ulrich, C. M. (2006) In silico experimentation with a model of hepatic ods Enzymol. 350, 87–96 mitochondrial folate metabolism. Theor. Biol. Med. Model. 3, 40 22. Wagner, W., Breksa, A. P., 3rd, Monzingo, A. F., Appling, D. R., and Rober- 38. Seither, R. L., Trent, D. F., Mikulecky, D. C., Rape, T. J., and Goldman, I. D. tus, J. D. (2005) Kinetic and structural analysis of active site mutants of (1989) Folate-pool interconversions and inhibition of biosynthetic pro- monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehy- cesses after exposure of L1210 leukemia cells to antifolates: experimental drogenase from Saccharomyces cerevisiae. Biochemistry 44, 13163–13171 and network thermodynamic analyses of the role of dihydrofolate poly- 23. Palmer, K. F., and Williams, D. (1974) Optical properties of water in the glutamates in antifolate action in cells. J. Biol. Chem. 264, 17016–17023 near infrared. J. Opt. Soc. Am. 64, 1107–1110 39. Horne, D. W., Patterson, D., and Cook, R. (1989) Effect of nitrous oxide 24. Barlowe, C. K., and Appling, D. R. (1990) Isolation and characterization of inactivation of vitamin B -dependent methionine synthetase on the sub- a novel eukaryotic monofunctional NAD -dependent 5,10-methylenetet- cellular distribution of folate coenzymes in rat liver. Arch. Biochem. Bio- rahydrofolate dehydrogenase. Biochemistry 29, 7089–7094 phys. 270, 729–733 25. Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, 40. Eto, I., and Krumdieck, C. L. (1982) Determination of three different pools T. L. (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134 of reduced one-carbon-substituted folates. III. Reversed-phase high-per- 26. Willems, E., Mateizel, I., Kemp, C., Cauffman, G., Sermon, K., and Leyns, formance liquid chromatography of the azo dye derivatives of p-amino- L. (2006) Selection of reference genes in mouse embryos and in differen- benzoylpoly--glutamates and its application to the study of unlabeled tiating human and mouse ES cells. Int. J. Dev. Biol. 50, 627–635 endogenous pteroylpolyglutamates of rat liver. Anal. Biochem. 120, 27. Kouadjo, K. E., Nishida, Y., Cadrin-Girard, J. F., Yoshioka, M., and St- 323–329 Amand, J. (2007) Housekeeping and tissue-specific genes in mouse tissues. 41. Veech, R. L. (1987) Pyridine nucleotides and control of metabolic pro- BMC Genomics 8, 127 cesses, in Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and 15516 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 289 • NUMBER 22 • MAY 30, 2014 MTHFD2L Exhibits Dual Redox Cofactor Specificity Medical Aspects, Part B (Dolphin, D., Poulson, R., and Avramovic, O., eds) lethality and neural tube and craniofacial defects in mice. Proc. Natl. Acad. pp. 79–105, John Wiley & Sons, New York Sci. U.S.A. 110, 549–554 42. Yang, H., Yang, T., Baur, J. A., Perez, E., Matsui, T., Carmona, J. J., Lam- 46. Alexiou, M., and Leese, H. J. (1992) Purine utilisation, de novo synthesis ming, D. W., Souza-Pinto, N. C., Bohr, V. A., Rosenzweig, A., de Cabo, R., and degradation in mouse preimplantation embryos. Development 114, Sauve, A. A., and Sinclair, D. A. (2007) Nutrient-sensitive mitochondrial 185–192 NAD levels dictate cell survival. Cell 130, 1095–1107 47. Albe, K. R., Butler, M. H., and Wright, B. E. (1990) Cellular concentrations 43. Sies, H. (1982) Nicotinamide nucleotide compartmentation, in Metabolic of enzymes and their substrates. J. Theor. Biol. 143, 163–195 Compartmentation (Sies, H., ed) pp. 205–231, Academic Press, New York 48. Corkey, B. E., Duszynski, J., Rich, T. L., Matschinsky, B., and Williamson, 44. Christensen, K. E., and MacKenzie, R. E. (2006) Mitochondrial one-car- J. R. (1986) Regulation of free and bound magnesium in rat hepatocytes bon metabolism is adapted to the specific needs of yeast, plants and mam- and isolated mitochondria. J. Biol. Chem. 261, 2567–2574 mals. Bioessays 28, 595–605 49. Saleet Jafri, M., and Kotulska, M. (2006) Modeling the mechanism of met- 45. Momb, J., Lewandowski, J. P., Bryant, J. D., Fitch, R., Surman, D. R., Vokes, abolic oscillations in ischemic cardiac myocytes. J. Theor. Biol. 242, S. A., and Appling, D. R. (2013) Deletion of Mthfd1l causes embryonic 801–817 MAY 30, 2014 • VOLUME 289 • NUMBER 22 JOURNAL OF BIOLOGICAL CHEMISTRY 15517

Journal

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

Published: May 30, 2014

Keywords: Folate Metabolism; Mitochondrial Metabolism; Multifunctional Enzymes; NAD; Neurodevelopment

There are no references for this article.