Decyanation of vitamin B12 by a trafficking chaperone

Abstract

The mystery of how the cyanide group in vitamin B(12) 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 B(12)-dependent target enzymes, methionine synthase and methylmalonyl-CoA mutase. Defects in MMACHC represent the most common cause of inborn errors of B(12) metabolism, and our results explain the observation that fibroblasts from these patients are poorly responsive to vitamin B(12) 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.

Fig. 1.

Structures of B₁₂ derivatives. (A) Structure of free base-on cobalamin showing that five of the six ligands are provided by the corrin ring. The lower axial ligand is the endogenous base, dimethylbenzimidazole. The upper axial ligand is variable, as indicated. MeCbl, AdoCbl, and H₂OCbl refer to methylcobalamin, deoxyadenosylcobalamin, and aquocobalamin respectively. (B) Conversion of vitamin B₁₂ to the active cofactor forms of B₁₂ requires a decyanation step. The product, cob(II)alamin, can be used for the synthesis of MeCbl or AdoCbl, which are bound in the base-off/His-on state in methionine synthase and methylmalonyl-CoA mutase, respectively. The differences in the UV-visible absorption spectra for these cobalamins lead to orange or red compounds as denoted.

Fig. 2.

Binding of CNCbl to MMACHC. (A) UV-visible absorption spectra of 10 μM free CNCbl (dashed line) or bound to 20 μM MMACHC (solid line) in 100 mM Hepes (pH 7.0), 150 mM KCl, and 5% glycerol. (Inset) Comparison between base-on (dashed line) and base-off (solid line) CNCbl in water and 6% HClO₄, respectively. (B) Binding of CNCbl to MMACHC was monitored by isothermal titration calorimetry of 25 μM MMACHC with CNCbl (1–500 μM) as described in Materials and Methods. Analysis of the titration data reveals stoichiometric binding and yields a K_d of 15.5 ± 1.1 μM.

Fig. 3.

Binding of methyl- or 5′-deoxyadenosylcobalamin to MMACHC. UV-visible absorption spectra of 10 μM free methyl- or 5′-deoxyadenosylcobalamin (dashed line) or bound to 20 μM MMACHC (solid line) in 100 mM Hepes (pH 7.0), 150 mM KCl, and 5% glycerol are shown.

Fig. 4.

Decyanation of CNCbl by MMACHC. (A) Spectrum of MMACHC reconstituted with CNCbl (blue) and its conversion to cob(II)alamin (red) in the presence of methionine synthase reductase and NADPH. (Inset) Rate of cob(II)alamin formation from 20 μM MMACHC/CNCbl in the presence of 4 μM methionine synthase reductase (●), 4 μM NR1 (▴), or in the absence of MMACHC (open symbols). (B) EPR spectra of cob(II)alamin (100 μM) generated in solution or formed by decyanation of 100 μM MMACHC/CNCbl as described in Materials and Methods.

Fig. 5.

Model for the decyanase activity of MMACHC. The dark blue domain in MMACHC represents the C-terminal TonB-like domain, which is proposed to open the “hatch” on the lysosomal transporter, in analogy with the role of TonB in bacterial two-component transport systems (6, 12).