The Structural Basis of Multifunctionality in a B12 Processing Enzyme

An early step in the intracellular processing of B12 involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B12) 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. A network of trafficking proteins tailor and escort B12, a reactive and rare cofactor, from its point of entry into the cell to its two client enzymes in mammals, methionine synthase and methylmalonyl-CoA mutase (1). An early step in this processing pathway involves accepting the B12 cargo as it exits the lysosome and converting the varied incoming derivatives into a common intermediate that can be partitioned for synthesis into the two biologically active forms, methylcobalamin (MeCbl) and 5 ́deoxyadenosylcobalamin (AdoCbl). This step is catalyzed by the CblC protein (2,3), the product of the cblC locus, a hotbed of mutations in the group of inherited cobalamin disorders (4). CblC is unique among B12 proteins in being able to manipulate the cobalt-carbon bond in B12 in both a homolytic and heterolytic fashion. It catalyzes the reductive decyanation of cyanocobalamin (CNCbl or vitamin B12) in the presence of a flavoprotein oxidoreductase (3). Remarkably, when presented with an alkylcobalamin, CblC also catalyzes the nucleophilic displacement of the alkyl group by glutathione (2) (Fig 1a). The lack of obvious sequence similarity between CblC and any protein of known structure has limited mechanistic insights into the structural basis of its remarkable chemical versatility. In this study, we report the crystal structures of human CblC in the apo(2.0 Å) and B12-bound (1.95 Å) forms, which reveal the presence of a large cavity for housing B12 and an N-terminal flavodoxin nitroreductase domain. The latter suggested that CblC might be a flavindependent B12 processing enzyme, which was confirmed by the demonstration that CblC catalyzes decyanation of CNCbl in the presence of FADH2. The structures provide a framework for understanding the biochemical penalties associated with patient mutations leading to methylmalonic aciduria and homocystinuria.