Coordination chemistry controls the thiol oxidase activity of the B12-trafficking protein CblC

Zhu Li; Aranganathan Shanmuganathan; Markus Ruetz; Kazuhiro Yamada; Nicholas A. Lesniak; Bernhard Kräutler; Thomas C. Brunold; Markos Koutmos; Ruma Banerjee

doi:10.1074/jbc.M117.788554

Coordination chemistry controls the thiol oxidase activity of the B₁₂-trafficking protein CblC

From the ^‡Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0600,
the ^§Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814,
the ^¶Institute of Organic Chemistry and Centre of Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria, and
the ^‖Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706

↵1 To whom correspondence should be addressed. Tel.: 734-615-5238; E-mail: rbanerje{at}umich.edu.

Edited by F. Peter Guengerich

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.

Footnotes

This work was supported in part by National Institutes of Health Grant DK45776 (to R. B.) and by American Heart Association Grant 13SDG14560056 (to M. K.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
The atomic coordinates and structure factors (code 5UJC) have been deposited in the Protein Data Bank (http://wwpdb.org/).
This article contains supplemental Table S1 and Figs. S1–S8.