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 oxidati ve inactivation. In bacteria, an NADPH-dependent two-protein system compris ing flavodoxin reductase and flavodoxin, transfers electrons during reactiv ation of methionine synthase. Until recently, the physiological reducing sy stem in mammals was unknown. Identification of mutations in the gene encodi ng a putative methionine synthase reductase in the cblE class of patients w ith an isolated functional deficiency of methionine synthase suggested a ro le for this protein in activation (Leclerc, D., Wilson, A., Dumas, R., Gafu ik, 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 enco ding human methionine synthase reductase and demonstrate that it is suffici ent for supporting NADPH-dependent activity of methionine synthase at a lev el that is comparable with that seen in the in vitro assay that utilizes ar tificial reductants. Methionine synthase reductase is a soluble, monomeric protein with a molecular mass of 78 kDa. It is a member of the family of du al flavoproteins and is isolated with an equimolar concentration of FAD and FMN. Reduction by NADPH results in the formation of an air stable semiquin one similar to that observed with cytochrome P-450 reductase. Methionine sy nthase reductase reduces cytochrome c in an NADPH-dependent reaction at a r ate (0.44 mu mol min(-1) mg(-1) at 25 degreesC) that is comparable with tha t reported for NR1, a soluble dual flavoprotein of unknown function, but is similar to 100-fold slower than that of P-450 reductase. The K-m for NADPH is 2.6 +/- 0.5 muM, and the K-act for methionine synthase reductase is 80. 7 +/- 13.7 nM for NADPH-dependent activity of methionine synthase.