Navigating the B(12) road: assimilation, delivery, and disorders of cobalamin

Abstract

The reactivity of the cobalt-carbon bond in cobalamins is the key to their chemical versatility, supporting both methyl transfer and isomerization reactions. During evolution of higher eukaryotes that utilize vitamin B12, the high reactivity of the cofactor coupled with its low abundance pressured development of an efficient system for uptake, assimilation, and delivery of the cofactor to client B12-dependent enzymes. Although most proteins suspected to be involved in B12 trafficking were discovered by 2009, the recent identification of a new protein reveals that the quest for elucidating the intracellular B12 highway is still far from complete. Herein, we review the biochemistry of cobalamin trafficking.

Keywords: Adenosylcobalamin; Cofactors; Metabolic Diseases; Metal Homeostasis; Trafficking; Vitamins.

FIGURE 1.

Cobalamin structure and conformations. a, cobalamin is shown in the base-on conformation, with DMB coordinating cobalt from the lower axial face of the corrin ring. The variable upper ligands are denoted as R on the right. b, alternative conformations of cobalamins differing with respect to the lower or α-axial ligation site.

FIGURE 2.

Model for cobalamin utilization and intracellular trafficking in mammals. Dietary cobalamin (R-Cbl) is bound by a series of proteins found in the saliva and stomach (haptocorrin (HC)), in the duodenum (intrinsic factor (IF)), and in blood (transcobalamin (TC)). The details of these early trafficking steps are not shown in this figure. Transcobalamin is recognized by the transcobalamin receptor (TCR) found on cell surfaces and endocytosed into the lysosome, where R-Cbl is released following proteolytic digestion of transcobalamin. R-Cbl exits the lysosome in a process that requires the products of the cblJ and cblF loci. In the cytoplasm, CblC converts R-Cbl (and CNCbl) to cob(II)alamin (Cbl²⁺), which is partitioned to (i) the MeCbl pathway in the cytoplasm, which involves CblG (methionine synthase), CblE (methionine synthase reductase), and CblD, and (ii) the AdoCbl pathway in the mitochondrion, which requires CblD, CblB (ATR), and CblA (G-protein or MeaB in bacteria) in addition to MCM (Mut). The question mark by the mitochondrial cobalamin transporter denotes that it is unidentified. M-CoA, methylmalonyl-CoA; S-CoA, succinyl-CoA.

FIGURE 3.

Biochemical functions of CblC and CblD. a, the structure of human CblC with MeCbl (Protein Data Bank code 3SC0). MeCbl (red) is bound in a base-off conformation, with the DMB tail located in a side pocket. The flavin reductase domain is shown in yellow. The arginine residues that are mutated in patients are shown in stick representation. b, reactions catalyzed by CblC. c, localization of mutations in CblD that lead to impaired AdoCbl or MeCbl synthesis and the minimal length required for binding to CblC. MLS, mitochondrial leader sequence.

FIGURE 4.

Cofactor loading, activity, and repair of mammalian cobalamin-dependent enzymes. a, methionine synthase (MS; CblG; blue square) catalyzes the overall transfer of a methyl group from N⁵-methyltetrahydrofolate to homocysteine to give methionine and tetrahydrofolate (THF). Occasional oxidative escape of the cob(I)alamin intermediate during the catalytic cycle leads to the inactive cob(II)alamin species. The latter is rescued to MeCbl in a reductive methylation reaction needing NADPH, methionine synthase reductase (MSR; CblE), and AdoMet. This repair reaction is also likely to represent the route for formation of MeCbl following transfer of cob(II)alamin to apomethionine synthase. The mechanism for cob(II)alamin transfer during methionine synthase reconstitution is not known. AdoHyc, S-adenosylhomocysteine. b, ATR (CblB; blue wheel) converts ATP and cob(II)alamin in the presence of a reductant to AdoCbl. Two equivalents of AdoCbl are bound at one time, and binding of ATP to the vacant site triggers transfer of one AdoCbl to the MCM (Mut; green circle) active site in a reaction that is gated by GTP hydrolysis in the G-protein chaperone (MeaB or CblA; orange rectangle). The GTP-bound state of MeaB blocks transfer of cob(II)alamin from ATR to the complex between MCM and G-protein. During catalysis, the cobalt-carbon bond is cleaved homolytically to initiate a radical-based mechanism for the conversion of methylmalonyl-CoA (M-CoA) to succinyl-CoA (S-CoA). Occasional loss of 5′-deoxyadenosine (Ado) from the active site precludes re-formation of AdoCbl at the end of the catalytic cycle and leads to inactive mutase. In this situation, the GTP-containing chaperone promotes dissociation of cob(II)alamin, permitting reconstitution of the mutase with active cofactor.