Structural Basis of Multifunctionality in a Vitamin B12-processing Enzyme

Abstract

An early step in the intracellular processing of vitamin B 12 involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B 12 ), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPHdependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.

Proteomics of vitamin B12 processing

“…Studies with the bovine isoform of MMACHC revealed that the reduced form of glutathione stabilizes MMACHC, suggesting that intracellular redox control could play a role in the regulation of the protein ' s lifetime [ 41 -44 ]. Koutmos [ 3 ] and Froese [ 45 ] glutathione-binding pocket up above the β -axial ligand position [ 45 ]. Importantly, the arginine-rich pocket comprises residues Arg161, Arg206 and Arg230 ( recombinant mutant Arg206Gln was insoluble suggesting a structural role for this residue, and that mutants Arg161Gln and Arg230Gln abolished GSH binding and dealkylase activity, which is a strong indication that these amino acid residues are critical for GSH binding [ 45 ].…”

“…High mRNA levels were detected in fetal liver with lower levels being detected in spleen, lymph node, thymus and bone marrow, and no message was detected in peripheral blood leukocytes [ 34 ]. Work by Koutmos et al showed that a truncated form of cblC lacking the last 38 amino acid residues is predominantly expressed in most tissues [ 3 ]. Importantly, a comprehensive examination of the mitochondrion proteome identified MMACHC as one of its resident proteins [ 35 ].…”

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

“…Two CblC variants, a full-length (32 kDa) and a truncated (26 kDa) form, have been reported in human fibroblasts and in murine tissue (35,36). The formation of the truncated CblC form is attributed to a predicted splicing variant (35).…”

“…Two CblC variants, a full-length (32 kDa) and a truncated (26 kDa) form, have been reported in human fibroblasts and in murine tissue (35,36). The formation of the truncated CblC form is attributed to a predicted splicing variant (35). Although the prevalence of the truncated CblC form is not known, the presence of the C-terminal domain diminishes the stability of human CblC and is not required for its function (35).…”

“…Human CblC is a soluble protein in which the C-terminal ϳ40 amino acid residues appear to be a recent evolutionary addition and are predicted to be highly disordered (35). Two CblC variants, a full-length (32 kDa) and a truncated (26 kDa) form, have been reported in human fibroblasts and in murine tissue (35,36).…”