Human Methionine Synthase Reductase, a Soluble P-450 Reductase-like Dual Flavoprotein, Is Sufficient for NADPH-dependent Methionine Synthase Activation*

Methionine synthase is a key enzyme in the methionine cycle that catalyzes the transmethylation of homocysteine to methionine in a cobalamin-dependent reaction that utilizes methyltetrahydrofolate as a methyl group donor. Cob(I)alamin, a supernucleophilic form of the cofactor, is an intermediate in this reaction, and its reactivity renders the enzyme susceptible to oxidative inactivation. In bacteria, an NADPH-dependent two-protein system comprising flavodoxin reductase and flavodoxin, transfers electrons during reactivation of methionine synthase. Until recently, the physiological reducing system in mammals was unknown. Identification of mutations in the gene encoding a putative methionine synthase reductase in the cblE class of patients with an isolated functional deficiency of methionine synthase suggested a role for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafuik, C., Song, D., Watkins, D., Heng, H. H. Q., Rommens, J. M., Scherer, S. W., Rosenblatt, D. S., and Gravel, R. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3059–3064). In this study, we have cloned and expressed the cDNA encoding human methionine synthase reductase and demonstrate that it is sufficient for supporting NADPH-dependent activity of methionine synthase at a level that is comparable with that seen in thein vitro assay that utilizes artificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of dual flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquinone similar to that observed with cytochrome P-450 reductase. Methionine synthase reductase reduces cytochrome c in an NADPH-dependent reaction at a rate (0.44 μmol min−1 mg−1 at 25 °C) that is comparable with that reported for NR1, a soluble dual flavoprotein of unknown function, but is ∼100-fold slower than that of P-450 reductase. TheK m for NADPH is 2.6 ± 0.5 μm, and the K act for methionine synthase reductase is 80.7 ± 13.7 nm for NADPH-dependent activity of methionine synthase.

Homocysteine is a key junction metabolite in the methionine cycle that is formed by the hydrolysis of S-adenosylhomocysteine, the product of S-adenosylmethionine-dependent methyl-ation reactions. Elevated levels of homocysteine are correlated with cardiovascular diseases, neural tube defects, and Alzheimer's disease (1)(2)(3)(4). Intracellular levels of homocysteine are kept low by the action of two classes of reactions. The transmethylation reactions catalyzed by methionine synthase or betaine homocysteine methyltransferase salvage homocysteine back to the methionine cycle. Alternatively, the ␤-replacement reaction catalyzed by cystathionine ␤-synthase commits homocysteine to the trans sulfuration pathway and provides a mechanism for generating cysteine from the essential amino acid, methionine. Of the two transmethylases, betaine homocysteine methyltransferase has a relatively restricted tissue distribution, whereas methionine synthase is believed to be ubiquitous (5).
The mammalian methionine synthase is a methylcobalamindependent enzyme that catalyzes the successive transfer of a methyl group from CH 3 -H 4 folate 1 to the cob(I)alamin form of the cofactor and from methylcobalamin to homocysteine to form methionine and tetrahydrofolate as products ( Fig. 1) (6). The reactivity of the intermediate cob(I)alamin form of the enzyme renders the enzyme susceptible to oxidation, resulting in adventitious formation of cob(II)alamin. The latter represents an inactive form of the enzyme that can be returned to the catalytic cycle by a reductive methylation reaction that utilizes S-adenosylmethionine as a methyl group donor. In bacteria, the physiological reducing system is comprised of two flavoproteins, NADPH-flavodoxin (or ferredoxin) oxidoreductase and flavodoxin, that relay electrons from NADPH to methionine synthase (Fig. 2) (7)(8)(9). Mammals lack flavodoxin, and until recently the physiological reducing system was completely unknown.
Three years ago, two studies, using genetic and biochemical approaches, respectively, identified two pathways for mammalian methionine synthase activation (Fig. 2). Using a homologybased PCR approach, Gravel and co-workers (10) were able to clone a soluble P-450-reductase-like protein and named it methionine-synthase reductase. The predicted amino acid sequence from the cDNA is homologous at the N terminus to the flavodoxin-like FMN-containing domain and linked by a hinge region to an NADPH-flavodoxin oxidoreductase-like FAD and a NADPH-binding domain found in cytochrome P-450 reductase. The sole but compelling evidence for the relevance of this putative protein in methionine synthase activation came from the identification of mutations in fibroblasts from cblE patients with functional methionine synthase deficiency (10,11). Biochemical and genetic characterization had earlier indicated * This work was supported by National Institutes of Health Grant DK45776. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Established Investigator of the American Heart Association. To whom correspondence should be addressed. that mutations in these cell lines were not in the gene encoding methionine synthase (which belong to the cblG complementation group) and were likely to be associated with the reductive activation system (12,13). However, biochemical characterization of the gene product and indeed its ability to activate methionine synthase were not investigated.
Biochemical investigations of the mammalian reductive activation system in our laboratory were based on reconstitution of the activation system in the presence of NADPH by combinations of different fractions from cell homogenates (13). Purification of one of these components resulted in its identification as soluble cytochrome b 5 and indicated that the second component was microsomal (14). This suggested the involvement of P-450 reductase and, indeed, methionine synthase activity could be reconstituted completely under in vitro conditions by the addition of purified P-450 reductase and soluble cytochrome b 5 .
These reports raised the following unresolved questions as depicted in Fig. 2. Can the putative methionine synthase reductase activate methionine synthase? Does soluble cytochrome b 5 play a role in methionine synthase reductase-mediated activation of methionine synthase? Is the cytochrome b 5 /P-450 reductase two-component activation system physiologically relevant, particularly because the proximal electron acceptor from NADPH, P-450 reductase, is microsomal, and the electron acceptor, methionine synthase, is soluble? As a first step toward answering these questions, we have cloned and expressed the human cDNA encoding methionine synthase reductase and characterized its basic properties. Our results demonstrate that methionine synthase reductase is a dual flavoprotein that is sufficient for supporting methionine synthase activity in the presence of NADPH.
Cloning the cDNA Encoding Human Methionine Synthase Reductase-The cDNA encoding methionine synthase reductase gene was cloned by PCR, using the following nondegenerate oligonucleotides based on the published sequence (10): forward, 5Ј-GCCTTGAATTCAT-GCGTCGTTTTCTGTTACTATATG-3Ј, and reverse, 5Ј-CTCGAGT-TATGACCAAATATCCTGAAG-3Ј. The residues in bold letters correspond to the EcoRI and XhoI restriction sites that were designed into the primer sequences. Total RNA was isolated from the human embryonal kidney cell line 293 (ATCC, Manassas, VA) using TRIZOL reagent (Life Technologies, Inc.), and cDNA was then synthesized by reverse transcriptase-PCR according to the vendor's protocol. Following amplification of this cDNA using Pfu polymerase (Stratagene), the gene was ligated into the TOPO-TA vector (Invitrogen) and sequenced to confirm that no PCR-induced errors had been introduced. The cDNA contained A and C nucleotides at positions 66 and 524, respectively, resulting in the amino acids Ile 22 and Ser 175 in the encoded protein. Ile 22 corresponds to a common polymorphism with an allele frequency of 0.49 (15). The frequency of Ser 175 is unknown. The conditions for PCR amplification were as follows: denaturation at 94°C for 30 s; followed by 35 cycles of 94°C for 15 s, 55°C for 30 s, and 68°C for 5 min; followed by extension at 68°C for 10 min. Subsequently, the 2.1-kilobase insert containing the open reading frame for methionine synthase reductase was excised using the EcoRI and XhoI restriction sites and subcloned in frame with the GST-coding sequence in the pGEX-4T1 vector (Amersham Pharmacia Biotech). The resulting plasmid, pHOMSR, was transformed into the Escherichia coli strain BL21(DE3) for expression studies.
Expression of Recombinant Human Methionine Synthase Reductase-The cells were grown overnight at 37°C in LB medium containing 100 g/ml ampicillin, and 10 ml of this culture was used to inoculate 1 liter of LB medium. The cultures were grown at 25°C to an A 600 of 0.6. Then isopropyl-1-thio-␤-D-galactopyranoside (1.0 mM) and riboflavin (1 mg/l) were added simultaneously, and the cultures were grown overnight. The cells were harvested by centrifugation at 5,000 ϫ g for 15 min and either stored at Ϫ80°C or processed immediately.
Purification Recombinant Human Methionine Synthase Reductase-The cell paste was resuspended in 200 ml of sonication buffer (50 mM Tris-HCl, pH 6.5, 10 mM EDTA, 0.01% lysozyme, 10 mM DTT, and protease inhibitors), and the cells were disrupted with a Sonicator XL2020 (Misonix Inc.) at a power setting of 7 (7 ϫ 40 s bursts alternating with 2-min pauses). The sonicate was centrifuged at 20,000 ϫ g for 1 h, and the supernatant was loaded onto a glutathione-Sepharose 4B column and washed with 1 liter of phosphate-buffered saline. Thrombin (400 units from Amersham Phamacia Biotech) treatment was subsequently performed overnight at room temperature on the column to release methionine synthase reductase from the fusion protein. The flow-through yielded relatively pure methionine synthase reductase, which was further purified by anion exchange chromatography on a 150 ϫ 25-mm POROS HQ-10 column (PerSeptive Biosystems) using a linear gradient ranging from 50 to 300 mM NaCl in 50 mM potassium phosphate buffer, pH 7.2, and a flow rate of 10 ml/min. Methionine synthase reductase-containing fractions were identified by their absorption at 280 and 450 nm and by Western blot analysis and concentrated by ultrafiltration. FMN (40 M) was added during concentration, and excess FMN was subsequently removed using a PD-10 (Amersham Pharmacia Biotech) gel filtration column. Protein concentrations were determined by Bradford analysis using reagents from Bio-Rad and bovine serum albumin as a standard.
Peptide Sequencing-The methionine synthase reductase protein band was excised from a 10% SDS-polyacrylamide gel and submitted to the Protein Core Facility (University of Nebraska, Lincoln, NE) for N-terminal sequencing on a Procise 494 instrument.
Flavin Determination and Spectral Analysis-The flavin cofactor content was determined by HPLC (16) using a fluorescence detector. Flavins were released by boiling methionine synthase reductase (60 g in 1 ml of 10 mM potassium phosphate buffer, pH 6.0) for 5 min and then cooling rapidly on ice. Denatured protein was removed by centrifugation at 20,000 ϫ g for 5 min. FAD and FMN were separated by injecting 30 l of the supernatant onto a C 18 reverse phase column (250 ϫ 4 mm, 5 micron particle size, fitted with a 4 ϫ 4-mm C 18 guard column from Beckman), using a gradient ranging from 80% 10 mM potassium phosphate, pH 6.0, 20% methanol to 50% 10 mM potassium phosphate, pH 6.0, 50% methanol at a flow rate of 1 ml/min. Flavins were detected by

FIG. 2.
Comparison of the reductive activation system for methionine synthase in E. coli versus alternative pathways that can be considered for the mammalian enzyme. Prior to this study, the ability of only the P-450 reductase/soluble cytochrome b 5 system to reconstitute methionine synthase activity had been demonstrated biochemically. In principle, methionine synthase reductase could function either independently or in the presence of soluble cytochrome b 5 to activate methionine synthase. The role, if any, of the dual flavoprotein, NR1, in this pathway is unknown. fluorescence (excitation, 450 nm; emission 520 nm). The column was calibrated using authentic FMN and FAD standards (Sigma), and the retention times were found to be 8.3 and 7.5 min, respectively.
Methionine Synthase Activation Assay-The NADPH-dependent methionine synthase assay was adapted from the published method (17,18) and monitors the transfer of [ 14 C]methyl group from the substrate, CH 3 -H 4 folate, to the product, methionine. The assay mixture contained 50 mM potassium phosphate buffer, pH 7.2, 100 mM potassium chloride, 500 M homocysteine, 19 M S-adenosylmethionine, 250 M (6-R,S)-5-[ 14 C]CH 3 -H 4 folate (ϳ2000 dpm/nmol), 25 mM DTT, 1 mM NADPH, and the indicated amounts of methionine synthase and methionine synthase reductase in a total volume of 1 ml. The mixture lacking CH 3 -H 4 folate was preincubated at 37°C for 5 min. The reaction was initiated with CH 3 -H 4 folate, incubated for 10 min at 37°C, and terminated by heating at 98°C for 2 min. The assay mixture was immediately placed on ice for 2 min and then passed through a small (0.5 ϫ 6 cm) column of Dowex 1X8-200 (chloride form). The column was washed with 2 ml of water, and the eluate was collected in a scintillation vial. Scintillation fluid (10 ml) was added to the aqueous sample, and the radioactivity was determined in a liquid scintillation counter. All reported values are corrected for the counts observed in control assays run in parallel from which methionine synthase reductase was omitted.
Ionic strengths were calculated as described by Perrin and Dempsey (19) to determine the concentration of KCl necessary to achieve the desired ionic strength in the chosen buffer. The ionic strength (I) of a solution is given by Equation 1, where c i is the concentration of each type of ion (in moles/liter), and z is its charge.
The ionic strength dependence of the standard in vitro assay in which DTT and B 12 replace NADPH and methionine synthase reductase was determined in 25 or 50 mM potassium phosphate buffer, pH 7.2, to which variable amounts of KCl were added to give ionic strengths ranging from 65 to 760 mM. The ionic strength dependence of the methionine synthase reductase/NADPH assay was determined in 50 mM potassium phosphate buffer, pH 7.2, to which KCl was added to give ionic strengths ranging from 120 to 820 mM. Cytochrome c Reduction Assay-Reduction of cytochrome c was monitored by measuring the absorbance change at 550 nm (⌬⑀ ϭ 21.1 mM Ϫ1 cm Ϫ1 ) (20). The reaction mixture contained 50 mM potassium phosphate buffer, pH 7.7, 60 M cytochrome c, 100 M NADPH, and methionine synthase reductase in a final volume of 1 ml. The reaction was initiated by addition of methionine synthase reductase, and initial velocities were determined at 25°C. The background rate, obtained in the absence of enzyme, was subtracted.
Purification of Methionine Synthase and Soluble Cytochrome b 5 -Porcine methionine synthase and soluble cytochrome b 5 were purified from pig liver as described previously (14,21). The specific activity of the porcine methionine synthase used in these studies was 1.8 mol min Ϫ1 mg Ϫ1 in the standard DTT/B 12 assay.
Generation of Methionine-Synthase Reductase Antibodies-To aid with the identification of the human enzyme during purification, antibodies were generated against the putative NADPH-binding domain. The expression vector was generated by PCR amplification and cloning of nucleotides 1596 -2093 (encoding amino acid residues 533-698) of methionine synthase reductase. This region was amplified using cDNA as a template from clone 704947 obtained from the IMAGE Consortium (Livermore, CA). Forward (5Ј-CGGATCCCCAGACGACCCTTCAATC-3Ј) and reverse (5Ј-CTCGAGTTATGACCAAATATCCTGAAG-3Ј) primers were used that contained BamHI and XhoI sites, respectively, for subcloning (in bold type). This amplicon was first ligated into the TOPO-TA vector, and its nucleotide sequence was determined to verify the absence of PCR-introduced sequence errors. The insert encoding the NADPH domain was subcloned in frame with the GST coding sequence in the pGEX-4T1 vector and expressed as a GST fusion protein in the E. coli strain BL21(DE3). The cells were grown and disrupted using the same conditions described above for the expression of full-length methionine synthase reductase. Most of the fusion protein was found in inclusion bodies that were solubilized in 6 M urea and purified by cation exchange chromatography on a CM-cellulose column using a gradient of NaCl in 50 mM Tris acetate, pH 4.5, ranging from 0 to 500 mM. Fractions containing the fusion protein were run on a 10% SDS-polyacrylamide gel, and the band of interest was excised and submitted to Alpha Diagnostic International Inc. (San Antonio, TX) for antibody generation in rabbits.

Purification of Recombinant Human Methionine Synthase
Reductase-Recombinant human methionine synthase reductase was expressed heterologously in E. coli as a fusion protein with GST. A two-step purification protocol yielded methionine synthase reductase with a final purity of Ͼ95% (Fig. 3). The estimated subunit molecular mass of the protein is 78 kDa as judged by SDS-polyacrylamide gel electrophoresis. This is consistent with the molecular mass of 77.7 kDa predicted from the open reading frame (10). Size exclusion chromatography yielded an estimate of 77 kDa for the native protein (not shown), indicating that methionine synthase reductase is monomeric. The identity of the recombinant protein was confirmed by N-terminal peptide sequence determination, which yielded the following sequence: GSPEFMRRFL. This exactly matches the predicted sequence for the first five residues of methionine synthase reductase (MRRFL) (10), preceded by the four amino acids following the thrombin recognition site in pGEX-4T1 (GSPEF).
Characterization of the Flavin Content of Methionine Synthase Reductase-Purified methionine synthase reductase is yellow, as expected for a flavoprotein, and in trial experiments bound to 2Ј,5Ј-ADP-agarose, an affinity matrix that is commonly used for purifying pyridine nucleotide-binding proteins (data not shown). HPLC analysis of heat-denatured enzyme revealed the presence of two fluorophores whose retention times matched exactly those of authentic FMN and FAD (not shown). Based on this analysis, 0.9 and 1.1 mol each of FMN and FAD, respectively, were found per mole of methionine synthase reductase.
The UV-visible spectrum of oxidized methionine synthase reductase is typical of a flavoprotein, with absorption maxima at 380 and 454 nm and a shoulder at 480 nm, similar to the spectrum reported for P-450 reductase (1-3) (Fig. 4). Addition of NADPH under aerobic conditions resulted in the appearance of a long wavelength absorption band with a peak at 585 nm and a shoulder at 640 nm. This is consistent with the formation of an air stable semiquinone as seen in P-450 reductase (22,23). The flavin concentration estimated from the absorption at 454 nm by assuming a molar absorptivity of 10.8 ϫ 10 3 M Ϫ1 cm Ϫ1 (24) yields an estimate of 2 mol flavin/mol methionine synthase reductase, consistent with the HPLC analysis. The extinction coefficient used here is similar to the value obtained from an equimolar mixture of FAD and FMN with ⑀450 nm ϭ of 11.3 ϫ 10 3 M Ϫ1 cm Ϫ1 (25) and 12.2 ϫ 10 3 M Ϫ1 cm Ϫ1 (26), respectively, and that from an equimolar mixture of the independently expressed flavin containing domains of human cytochrome P-450 reductase (27).
Methionine Synthase Reductase Activity-The activity of porcine methionine synthase in the presence of NADPH and recombinant methionine synthase reductase is comparable with that observed in the standard in vitro assay containing DTT and B 12 (see Table II). No activity is observed in the absence of methionine synthase reductase in the NADPH assay. Because previous biochemical studies on the physiological reductive activation system of methionine synthase had indicated the presence of two components (13) and led to the isolation of soluble cytochrome b 5 (14), the effect of the latter on the methionine synthase reductase-dependent activity was examined. As can be seen from the data in Table II, the presence of cytochrome b 5 had no effect on this assay, indicating that methionine synthase reductase is sufficient for reconstitution of methionine synthase activity in the presence of NADPH.
Because protein-protein interactions are involved in the methionine synthase reactivation reaction, the ionic strength dependence of the assay was examined (Fig. 5). Surprisingly, the activity of methionine synthase in the standard assay in which DTT and B 12 serve to reduce the enzyme also shows an ionic strength dependence that has not been previously reported to our knowledge. The maximal activity plateaus between ionic strengths ranging from 300 to 600 mM. In contrast, the methionine synthase reductase-dependent assay displays a different ionic strength dependence. Maximal activity is observed at ϳ220 mM, and higher ionic strength leads to decreased activity.
Both NADPH and NADH can support methionine synthase activity, albeit with very different efficacies (Fig. 6). The preferred electron donor is NADPH, with an apparent K m of 2.64 Ϯ 0.54 M. NADH can replace NADPH but only at significantly higher and nonphysiological concentrations. Reductive activation of methionine synthase by methionine synthase reductase displays saturation kinetics (Fig. 7). Kinetic analysis of the data yielded a K act value for methionine synthase reductase of 80.7 Ϯ 13.7 nM and a V max value of 2.14 Ϯ 0.12 mol min Ϫ1 mg Ϫ1 . Maximal activity of methionine synthase was observed at a stoichiometry of ϳ4 mol of methionine synthase reductase/ mol of methionine synthase.
Cytochrome c Reductase Activity of Methionine Synthase Reductase-Like cytochrome P-450 reductase, methionine synthase reductase is able to reduce the hemeprotein cytochrome c (not shown). The cytochrome c reductase activity of methionine synthase reductase (V max ϭ 0.44 mol min Ϫ1 mg Ϫ1 protein) is, however, significantly lower (1%) than that of cytochrome P-450 reductase but comparable with that of a newly described and homologous flavoprotein of unknown function, NR1 (28). DISCUSSION Functional deficiency of methionine synthase is inherited as an autosomal recessive disorder. It is accompanied by pleiotropic clinical presentations including hyperhomocysteinemia with attendant severe cardiovascular problems, hypomethioninemia, megaloblastic anemia, and developmental delay (29). In principle, an isolated functional deficiency of methionine synthase could arise from defects in the enzyme itself or in the reductive activation system (30). Consistent with this expectation, two distinct genetic complementation groups have been recognized in patients: cblG with defects in methionine synthase and cblE with defects in the reductive activation system (12). This has been confirmed by the recent cloning and FIG. 5. Ionic strength dependence of the standard and methionine synthase reductase dependent assays. Activity of methionine synthase in the standard assay containing DTT and B 12 increases linearly at low ionic strength and plateaus between 300 and 600 mM (q). In contrast, the activity of methionine synthase in the NADPH/ methionine synthase reductase assay is maximal at 220 mM and decreases at higher ionic strengths (E). mutation identification in the genes encoding methionine synthase (31,32) and methionine synthase reductase (10,11), respectively. However, the possibility of a third complementation group has been raised recently based on characterization of a patient cell line with functional methionine synthase deficiency (33). This raises the possibility that additional or alternative components may be involved in the function of methionine synthase in vivo.
Studies with patient cell lines are directly relevant to the issue of the nature of the physiological reducing system for the mammalian methionine synthase. The recent description of methionine synthase reductase (10) and the soluble cytochrome b 5 /P-450 reductase system (14) point to the involvement of two related mammalian proteins, which represent the evolutionary fusion product of the bacterial reductive activation system, flavodoxin, and NADPH-flavodoxin oxidoreductase (34,35). However, they also raise several questions, in particular, whether or not a unique reductive activation system for methionine synthase exists in mammals and whether the soluble cytochrome b 5 /P-450 reductase system is physiologically relevant. Methionine synthase has been shown to be an essential gene in mice and a homozygous knockout of this gene results in embryonic lethality (36). Hence the stakes are high for maintaining active methionine synthase. The reported existence of functionally null mutations in patients with the cblG variant form of methionine synthase deficiency (37) is controversial, because low levels of methionine synthase activity have been measured in some of these cell lines (13). This discrepancy may be explained by the possibility that normal splicing occurs at a low frequency giving rise to very low levels of wild type enzyme because one allele in each of these cell lines carries a mutation that is expected to unmask cryptic splice sites leading to functionally null phenotypes.
As a first step toward resolving the many open questions regarding the components of the physiological reducing system of mammalian methionine synthase, we have cloned, expressed, and characterized the putative human methionine synthase reductase. The latter is one of a small number of dual flavoproteins, a family that includes cytochrome P-450 reductase (22), nitric oxide synthase (38,39), sulfite reductase (40), and NR1 (28). It is 38% identical to the human microsomal cytochrome P-450 reductase and is predicted to have an N-terminal FMN-binding domain and a C-terminal FAD/ NADPH-binding domain separated by a linker region. It is 41% identical to NR1, a soluble dual flavoprotein of unknown function (28). The predicted multidomainal organization of methionine synthase reductase is also suggested by its proteolytic lability during purification that results in the generation of two fragments (not shown) and contributes to the low yield following the limited proteolysis step (Table I).
Recombinant human methionine synthase reductase is isolated with an equimolar content of FAD and FMN and an absorption spectrum that is consistent with the presence of fully oxidized flavin (Fig. 4). Reduction with NADPH under aerobic conditions results in the appearance of long wavelength absorption consistent with the formation of an air stable semiquinone as seen with P-450 reductase. Methionine synthase reductase catalyzes the reduction of cytochrome c, with a specific activity that is comparable with that of NR1 (28) but ϳ100-fold lower than that of P-450 reductase (41).
In contrast to the two component P-450 reductase/soluble cytochrome b 5 system, methionine synthase reductase alone is capable of fully supporting NADPH-dependent activity of methionine synthase under in vitro conditions (Table II). Indeed, the addition of soluble cytochrome b 5 has no effect on this activity. This result excludes one of the pathways that had been considered earlier (42) by demonstrating that, at least under in vitro conditions, cytochrome b 5 plays no role in methionine synthase reductase-dependent activation of methionine synthase (Fig. 2).
Extensive structure function studies of the E. coli methionine synthase suggest that major domain movements accompany the transition from the catalytically active to inactive state (43). Thus, under turnover conditions, the N-terminal substrate binding domains must interact with the central B 12binding domain, whereas under activation conditions, the C-terminal S-adenosylmethionine-binding domain, flavodoxin, and the B 12 -binding domain form a complex. It is therefore not surprising that the activity of methionine synthase, even in the absence of a physiological reducing partner, shows an ionic strength dependence (Fig. 5). The different conformations are expected to involve surface interactions between the respective domains, and indeed cross-linking and modeling studies have revealed the importance of surfacial electrostatic interactions in the activation conformation (43). However, the ionic strength FIG. 7. Dependence of the NADPH-dependent methionine synthase activity on the concentration of methionine synthase reductase. The concentrations of methionine synthase and NADPH employed in these experiments were 26 nM and 1 mM, respectively. The experimental data were fitted to the Michaelis-Menten equation and yielded a K act value for MSR of 80.7 Ϯ 13.7 nM and a V max value of 2.14 Ϯ 0.12 mol min Ϫ1 mg Ϫ1 . The inset shows determination of the stoichiometry of MSR to MS (methionine synthase) that is required for full activation. dependence of methionine synthase in the presence of methionine synthase reductase is distinct from that in the presence of exogenous reductants such as DTT and B 12 and displays a narrow range within which activity is optimal. At physiologically relevant concentrations of the reduced pyridine nucleotides, methionine synthase reductase is expected to utilize NADPH almost exclusively (Fig. 6). The K m for NADPH (2.6 Ϯ 0.5 M) is comparable with that reported for recombinant rat liver cytochrome P-450 reductase (6.2 Ϯ 0.7 M) (41). The methionine synthase reductase-dependent activity of methionine synthase shows saturation kinetics with a K act of methionine synthase reductase of 80.7 Ϯ 13.7 nM. This is higher than the K m for flavodoxin (ϳ5 nM) for the E. coli methionine synthase (7). Maximal activity is observed at a stoichiometry of ϳ4 methionine synthase reductase/methionine synthase, suggesting that the binding interactions between these two proteins is weaker than that of the corresponding redox pair in E. coli, where a ratio of 1 has been reported (7).
In summary, these studies report on the expression and characterization of recombinant human methionine synthase reductase, a function that has only been deduced previously based on its predicted sequence and identification of mutations in cblE patient cell lines. We demonstrate that methionine synthase reductase is fully capable of supporting methionine synthase activity in the presence of NADPH and that it is a member of the growing family of dual flavoproteins. The issue of whether or not methionine synthase reductase represents the sole physiologically relevant pathway for activation of methionine synthase remains open with other candidate pathways remaining to be evaluated. Ϫ methionine synthase reductase 0 ϩ methionine synthase reductase 1.7 ϩ methionine synthase reductase ϩ soluble cytochrome b 5

1.66
Standard assay (ϩ DTT ϩ B 12 ) 1.8 a The NADPH-dependent assay described under "Experimental Procedures" was employed for the first three entries, and the assay mixtures each contained the same amount of porcine methionine synthase. In the standard assay, NADPH and methionine synthase reductase were replaced by DTT and B 12 as described under "Experimental Procedures."