Abstract
The cobalamin or B12 cofactor supports sulfur and one-carbon metabolism and the catabolism of odd-chain fatty acids, branched-chain amino acids, and cholesterol. CblC is a B12-processing enzyme involved in an early cytoplasmic step in the cofactor-trafficking pathway. It catalyzes the glutathione (GSH)-dependent dealkylation of alkylcobalamins and the reductive decyanation of cyanocobalamin. CblC from Caenorhabditis elegans (ceCblC) also exhibits a robust thiol oxidase activity, converting reduced GSH to oxidized GSSG with concomitant scrubbing of ambient dissolved O2 The mechanism of thiol oxidation catalyzed by ceCblC is not known. In this study, we demonstrate that novel coordination chemistry accessible to ceCblC-bound cobalamin supports its thiol oxidase activity via a glutathionyl-cobalamin intermediate. Deglutathionylation of glutathionyl-cobalamin by a second molecule of GSH yields GSSG. The crystal structure of ceCblC provides insights into how architectural differences at the α- and β-faces of cobalamin promote the thiol oxidase activity of ceCblC but mute it in wild-type human CblC. The R161G and R161Q mutations in human CblC unmask its latent thiol oxidase activity and are correlated with increased cellular oxidative stress disease. In summary, we have uncovered key architectural features in the cobalamin-binding pocket that support unusual cob(II)alamin coordination chemistry and enable the thiol oxidase activity of ceCblC.
Highlights
Thiol oxidase activity of ceCblCA combination of kinetic, spectroscopic, computational, and crystallographic approaches has revealed the involvement of unusual cobalt coordination chemistry that is supported by the active-site architecture of ceCblC but not hCblC
2.9 Ϯ 0.1 19 Ϯ 2 21 Ϯ 2 83 Ϯ 9 102 Ϯ 9 a Cobalamin processing rates were determined by mixing ceCblC (40 M)-cobalamin (20 M) with 4 mM GSH in Buffer A under anaerobic conditions at 20 °C
(0.5–5 M) complex in the presence of 4 mM GSH under aerobic conditions. c From Ref. 12. d From Ref. 13. e ceCblC-OH2Cbl complex was generated by oxidation of ceCblC-cob(II)alamin in the presence of the xanthine/xanthine oxidase system followed by rigorous washing to remove xanthine
Summary
A combination of kinetic, spectroscopic, computational, and crystallographic approaches has revealed the involvement of unusual cobalt coordination chemistry that is supported by the active-site architecture of ceCblC but not hCblC. These studies support a chemical mechanism of thiol oxidation in which glutathionyl-cobalamin (GSCbl) is formed as an intermediate and GSH-dependent deglutathionylation of GSCbl yields GSSG
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