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ABSTRACT: Pathways for tailoring and processing vitamins into active cofactor forms exist in mammals that are unable to synthesize these cofactors de novo. A prerequisite for intracellular tailoring of alkylcobalamins entering from the circulation is removal of the alkyl group to generate an intermediate that can subsequently be converted into the active cofactor forms. MMACHC, a cytosolic cobalamin trafficking chaperone, has been shown recently to catalyze a reductive decyanation reaction when it encounters cyanocobalamin. In this study, we demonstrate that this versatile protein catalyzes an entirely different chemical reaction with alkylcobalamins using the thiolate of glutathione for nucleophilic displacement to generate cob(I)alamin and the corresponding glutathione thioether. Biologically relevant thiols, e.g. cysteine and homocysteine, cannot substitute for glutathione. The catalytic turnover numbers for the dealkylation of methylcobalamin and 5'-deoxyadenosylcobalamin by MMACHC are 11.7 +/- 0.2 and 0.174 +/- 0.006 h(-1) at 20 degrees C, respectively. This glutathione transferase activity of MMACHC is reminiscent of the methyltransferase chemistry catalyzed by the vitamin B(12)-dependent methionine synthase and is impaired in the cblC group of inborn errors of cobalamin disorders.
Journal of Biological Chemistry 10/2009; 284(48):33418-24. · 4.77 Impact Factor
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ABSTRACT: The MMACHC gene product of the cblC complementation group, referred to as the cblC protein, catalyzes the in vitro and in vivo decyanation of cyanocobalamin (vitamin B(12)). We hypothesized that the cblC protein would also catalyze the dealkylation of newly internalized methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl), the naturally occurring alkylcobalamins that are present in the diet. The hypothesis was tested in cultured endothelial cells using [(57)Co]-AdoCbl and MeCbl analogs consisting of [(57)Co]-labeled straight-chain alkylcobalamins ranging from C2 (ethylcobalamin) to C6 (hexylcobalamin). [(57)Co]-AdoCbl was converted to [(57)Co]-MeCbl by cultured bovine aortic endothelial cells, suggesting that a dealkylation process likely involving the cblC protein removed the 5'-deoxyadenosyl alkyl group. Surprisingly, all of the straight-chain alkylcobalamins served as substrates for the biosynthesis of both AdoCbl and MeCbl. Dealkylation was then assessed in normal skin fibroblasts and fibroblasts derived from three patients with mutations in the MMACHC gene. While normal skin fibroblasts readily converted [(57)Co]-propylcobalamin to [(57)Co]-AdoCbl and [(57)Co]-MeCbl, there was little or no conversion in cblC mutant fibroblasts. These studies suggest that the CblC protein is responsible for early processing of both CNCbl (decyanation) and alkylcobalamins (dealkylation) in mammalian cells.
Molecular Genetics and Metabolism 05/2009; 97(4):260-6. · 3.19 Impact Factor
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ABSTRACT: The mystery of how the cyanide group in vitamin B(12) or cyanocobalamin, discovered 60 years ago, is removed, has been solved by the demonstration that the trafficking chaperone, MMACHC, catalyzes a reductive decyanation reaction. Electrons transferred from NADPH via cytosolic flavoprotein oxidoreductases are used to cleave the cobalt-carbon bond with reductive elimination of the cyanide ligand. The product, cob(II)alamin, is a known substrate for assimilation into the active cofactor forms, methylcobalamin and 5'-deoxyadenosylcobalamin, and is bound in the "base-off" state that is needed by the two B(12)-dependent target enzymes, methionine synthase and methylmalonyl-CoA mutase. Defects in MMACHC represent the most common cause of inborn errors of B(12) metabolism, and our results explain the observation that fibroblasts from these patients are poorly responsive to vitamin B(12) but show some metabolic correction with aquocobalamin, a cofactor form lacking the cyanide ligand, which is mirrored by patients showing poorer clinical responsiveness to cyano- versus aquocobalamin.
Proceedings of the National Academy of Sciences 10/2008; 105(38):14551-4. · 9.68 Impact Factor
