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

Horatiu Olteanu, Ruma Banerjee

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Abstract

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 the in 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. The Km for NADPH is 2.6 ± 0.5 μM, and the Kact for methionine synthase reductase is 80.7 ± 13.7 nM for NADPH-dependent activity of methionine synthase.

Original languageEnglish (US)
Pages (from-to)35558-35563
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number38
DOIs
StatePublished - Sep 21 2001

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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