A Human Vitamin B12 Trafficking Protein Uses Glutathione Transferase Activity for Processing Alkylcobalamins

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°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.

Proteomics of vitamin B12 processing

“…In addition, MMACHC was capable of dealkylating a series of MeCbl analogues namely, ethylcobalamin, propylcobalamin, butylcobalamin, pentylcobalamin and hexylcobalamin [ 37 ]. MMACHC catalyzed the removal of the alkyl group at the upper axial position of all of the MeCbl analogs, however, the rate of dealkylation decreased with increasing alkyl chain length [ 38 ]. Whether the latter is a result of conformational alterations in the MMACHC protein induced by the more bulky alkyl moieties or due to an unfavorable incorporation of the longer alkyl carbocations into glutathione remains to be elucidated.…”

“…Mechanistically, dealkylation of AdoCbl and MeCbl is distinct from the decyanation pathway. MMACHC catalyzes the dealkylation of alkylcobalamins by a reaction involving the nucleophilic attack of the CoϪC bond by the thiolate anion of glutathione [ 38 ]. Demethylation of MeCbl was much faster than the removal of the 5 ′ -adenosyl group from AdoCbl (11.7 ± 0.2 and 0.150 ± 0.006/h, respectively) [ 38 ].…”

“…MMACHC catalyzes the dealkylation of alkylcobalamins by a reaction involving the nucleophilic attack of the CoϪC bond by the thiolate anion of glutathione [ 38 ]. Demethylation of MeCbl was much faster than the removal of the 5 ′ -adenosyl group from AdoCbl (11.7 ± 0.2 and 0.150 ± 0.006/h, respectively) [ 38 ]. In addition, MMACHC was capable of dealkylating a series of MeCbl analogues namely, ethylcobalamin, propylcobalamin, butylcobalamin, pentylcobalamin and hexylcobalamin [ 37 ].…”

Protection of aquo/hydroxocobalamin from reduced glutathione by a B12 trafficking chaperone

“…hCblC binds cyanocobalamin (CNCbl), MeCbl and AdoCbl in the base-off states (8,9), in which the DMB ligand is dissociated from cobalt. The protein catalyzes the reductive elimination of the cyanide ligand from CNCbl (9) and the elimination of the alkyl ligands (the methyl and the 5'-deoxyadenosyl ligands of MeCbl and AdoCbl, respectively) using reduced glutathione (GSH) as the cosubstrate (10). The reaction products of decyanation and dealkylation are cob(II) balamin (Co 2+ Cbl) and cob(I)balamin (Co 1+ Cbl), respectively, under anaerobic conditions, which are extremely oxygen sensitive.…”

Novel Deletion Mutation Identified in a Patient with Late-Onset Combined Methylmalonic Acidemia and Homocystinuria, cblC Type

“…Although the exact function of the protein is currently unknown, it appears to play an important role in the synthesis of cobalamin intermediates. For instance, it has been shown that MMACHC can catalyze a reductive decyanation reaction of cyanocobalamin (CNCbl) to yield cob(II)alamin (Kim et al 2008) and a dealkylation reaction of AdoCbl or MeCbl using glutathione as an electron donor Kim et al 2009). Furthermore, MMACHC has been proposed to interact with combined methylmalonic aciduria and homocystinuria cblD type (MMADHC), the downstream protein in the cobalamin pathway (Deme et al 2012;Plesa et al 2011).…”