Cloning and characterization of methionine synthase interacting protein involved in mammalian methionine synthase reductive methylation pathway

Methionine synthase (MS), catalyzes two successive transmethylation reactions in the de novo synthesis of methionine. In one half reaction, the methyl group from 5-CH3-tetrahydrofolate (5-CH3 THF) is transferred to the MS bound cobalamin(I) cofactor; in the second half of the reaction, methyl group is then transferred to homocysteine to generate methionine. After multiple rounds of catalysis, the supernucleophilic cobalamin(I) cofactor is oxidized to cobalamin(II), rendering MS inactive. In order for MS to reenter the catalytic cycle, a reductive methylation system is required. In E. coli, two flavoproteins, flavodoxin:NADP+ oxidoreductase and flavodoxin, along with NADPH and S-adenosyl methionine (Ado-met) serve as the circuits to supply e-1 and methyl group that reductively methylates the cobalamin cofactor. Recently, two groups have reported the existence of analogous reductive methylation systems in mammals. Chen and Banerjee have reconstituted porcine MS activity with purified porcine soluble cytochrome b5 and microsomal NADPH P450 reductase using NADPH as the ultimate electron source in an anaerobic MS assay. Leclerc et al. have cloned a flavoprotein, methionine synthase reductase (MSR), that was found to be mutated in patients (complementation group cblE) with deficiency in the reductive methylation pathway.; MS can be separated into 4 distinct regions---homocysteine, 5-CH 3 THF, cobalamin, and Ado-met binding domains. Ado-met binding region is required for the reductive methylation pathway. In addition, binding of E. coli flavodoxin to cob(II)alamin bound MS is thought to be via at least two lysine residues (K1035 and K1188) in this region. These residues are conserved in human MS (hMS). In addition, another group of patients ( cblG) with defect in the reductive methylation pathway has a P1173L mutation in hMS further supports the hypothesis that this region physically interacts with the mammalian reductive activation partners.; Using the Ado-met binding region of hMS as bait in yeast two-hybrid system, we cloned a gene that was able to immunoprecipitate mammalian MS from cell extracts. This protein was named m&barbelow;ethionine s&barbelow;ynthase i&barbelow;nteracting p&barbelow;rotein (MSIP). Full-length cDNA of MSIP was obtained and its predicted open reading frame contained 611 amino acids, giving rise to a protein with predicted size of 68 kDa. Sequence alignment revealed that MSIP belongs to phosphoglucomutase (PGM)/phosphomannomutase (PMM) family; while it possesses no PGM/PMM activity. It was found that MSIP was able to reduce cytochrome C using NADPH as electron donor in vitro, with 1000 fold lower activity than NADPH P450 reductase. MSIP and MSR was shown to maintain mammalian MS to be in the cob(I)alamin bound active form as seen in MSIP and MSR over-expressing cell lines. MSIP with cytochrome C to a greater extent and with cytochrome b 5 to a less extent and MSR alone were all able to reconstitute porcine MS in vitro. However, interaction studies using co-immunoprecipitation technique failed to show interaction between MS and MSR; while MS interacts with cytochrome b5, and MSIP specifically. This evidence along with subcellular localization data and parallel spatial distribution of MSIP and hMS in liver and kidney, suggested that MSIP with a cytochrome-like protein represents an alternate mechanism of reductive activation for mammalian MS in vivo.