Biochemical studies on a SNP and Patient Mutations in Mitochondrial B12 Proteins

Abstract

Humans have evolved a sophisticated trafficking pathway for absorption and utilization of a reactive and less abundant cofactor B12. Methylmalonyl-CoA mutase (MCM) utilizes AdoCbl synthesized by the ATP:cobalamin adenosyltransferase (ATR) in the mitochondria to catalyze isomerization reaction of M-CoA to S-CoA crucial for odd-chain fatty acid and branch chain amino acid metabolism. S174L, R190C/H and E193K ATR patient mutation and R532H SNP variation in MCM were studied in efforts understand biochemistry of cobalamin trafficking in mitochondria. Impaired Arg190 mutant’s ability to synthesis AdoCbl suggested its likely involvement in catalyzing the adenosyl transfer reaction catalyzed by ATR. ATP in addition to serving as substrate promotes formation of cobalamin binding pocket, impairment in ATP binding pocket in R190C/H and E193K mutants possibly translats into formation of defective cobalamin binding pocket displaying weaker affinity towards cob(II)alamin. Arg190 mutants have conserved ATR active site property to show stronger affinity for AdoCbl in the presence of triphosphate, but without performing any chemistry on the Co-C bond unlike wild-type ATR, invoking the importance of triphosphate in the active site. R532H variation in MCM resulting higher serum B12 levels show a similar substrate binding and M-CoA turnover activity like the wild-type MCM, however physiological effects of slow AdoCbl transfer from ATR to MCM in R532H variation need to be understood. These biochemical studies in addition to helping us understand the cobalamin trafficking could also assist in development of effective treatment strategy for patients born with inborn errors of B12 metabolism.