Decyanation of vitamin B ₁₂ by a trafficking chaperone

Abstract

The mystery of how the cyanide group in vitamin B12 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 B12-dependent target enzymes, methionine synthase and methylmalonyl-CoA mutase. Defects in MMACHC represent the most common cause of inborn errors of B12 metabolism, and our results explain the observation that fibroblasts from these patients are poorly responsive to vitamin B12 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.cobalamin ͉ flavin oxidoreductase ͉ methylmalonic aciduria ͉ homocystinuria ͉ cyanide V itamin B 12 (or cyanocobalamin, CNCbl) was discovered exactly 60 years ago in the laboratories of Folkers and Smith as the anti-pernicious anemia factor (1, 2), and its structure was revealed a few years later by the crystallographic studies in the Hodgkin laboratory (3). B 12 is one of the most complex cofactors in nature and belongs to the family of tetrapyrrolic-derived macrocyclic compounds. Five of the six ligands to the central cobalt atom in free B 12 are endogenous, being provided by the corin ring itself (Fig. 1A). In CNCbl, the sixth ligand, which occupies the upper axial position, is a cyano group. Although the biological origin of the cyano group in vitamin B 12 is unknown, it is the cofactor form commonly used in multivitamin formulations and one that individuals on vitamin supplementation are regularly exposed to. Decyanation of CNCbl is a prerequisite for its conversion to the active cofactor forms used by mammals in which the cyano ligand is replaced by a methyl group (in methylcobalamin) or a 5Ј-deoxyadenosyl group (in coenzyme B 12 or 5Ј-deoxyadenosylcobalamin) (Fig. 1B).Defects in B 12 metabolism are inherited as autosomal recessive disorders and are classified as belonging to one of eight genetic complementation groups, cblA-G and mut (4). Two loci, cblG and mut, encode B 12 -dependent enzymes: methionine synthase, which is cytoplasmic, and methylmalonyl-CoA mutase, which is mitochondrial. The remaining loci encode functions needed for intercompartmental trafficking and assimilation of the cofactor into its active forms. B 12 is present at relatively low concentrations in human tissues (5) and is reactive in all three of its biologically relevant oxidation states, factors that pose challenges for its unescorted delivery to target enzymes (6). The problem is further exacerbated by the use of the ''base-off/Hison'' conformation of the cofactor ...

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity

“…Hence, it is possible that M. barkeri strain Fusaro and the Sporomusa isolates provided the D. mccartyi strains with not yet identified compounds that enhance growth yields. Another possible explanation for the lower growth yields observed with CN-Cbl is the cell's energetic burden associated with the replacement of the upper cyanide ligand [33].…”

Proteomics of vitamin B12 processing

“…The reductive decyanation of CNCbl is catalyzed by the MMACHC protein in the presence of a flavoprotein reductase and NADPH [ 36 ]. The authors reported that cblC bound both MeCbl and AdoCbl inducing their base-off conformation [ 36 ]; however, it did not catalyze the dealkylation of MeCbl and AdoCbl, the two major dietary forms of Cbl. This intriguing finding was re-examined via ex vivo studies [ 37 ], and a new function was uncovered for the cblC protein: MMACHC is also a Cbl dealkylase [ 37 ].…”

“…The mystery of how decyanation of cyanocobalamin (CNCbl) occurs was recently solved by the in vitro studies of Kim et al [ 36 ]. The reductive decyanation of CNCbl is catalyzed by the MMACHC protein in the presence of a flavoprotein reductase and NADPH [ 36 ].…”

Navigating the B12 Road: Assimilation, Delivery, and Disorders of Cobalamin

“…Similarly, in the CblC-catalyzed dealkylation reaction, the glutathione thioether forms upon transfer of the alkyl group in addition to cob(I)alamin. When CNCbl is in the active site, electrons provided by free or protein-bound reduced flavin promote reductive homolytic cleavage, leading to cyanide elimination (13,35). Based on UV-visible and EPR spectroscopy, baseoff cob(II)alamin has been identified as the other product of the reductive elimination, consistent with a homolytic cleavage reaction mechanism.…”

“…The identity of the upper or ␤-axial ligand varies and includes cyano, aquo, methyl, and 5Ј-deoxyadenosyl groups. Ironically, 6 decades after its discovery, the origin and biological relevance of the cyano group remain unknown, although we have recently described a decyanase activity in the processing pathway (13), which allows utilization of CNCbl used in vitamin supplements. As the cobalt oxidation state decreases from 3 ϩ 3 2 ϩ 3 1 ϩ , the coordination number typically decreases from 6 3 5 3 4.…”