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Unusual Aerobic Stabilization of Cob(I)alamin by a B12-trafficking Protein Allows Chemoenzymatic Synthesis of Organocobalamins Zhu Li, Nicholas A Lesniak, and Ruma Banerjee J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/ja5077316 • Publication Date (Web): 04 Nov 2014 Downloaded from http://pubs.acs.org on November 8, 2014

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Journal of the American Chemical Society

Unusual Aerobic Stabilization of Cob(I)alamin by a B12trafficking Protein Allows Chemoenzymatic Synthesis of Organocobalamins Zhu Li, Nicholas A. Lesniak and Ruma Banerjee* Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0600

Supporting Information Placeholder ABSTRACT: CblC, a B12 trafficking protein, exhibits glutathione transferase and reductive decyanase activities for processing alkylcobalamins and cyanocobalamin, respectively, to a common intermediate that is subsequently converted to the biologically active forms of the cofactor. We recently discovered that the Caenorhabditis elegans CblC catalyzes thiol-dependent decyanation of CNCbl and reduction of OH2Cbl and stabilizes the paramagnetic cob(II)alamin product under aerobic conditions. In this study, we report the striking ability of the worm CblC to stabilize the highly reactive cob(I)alamin product of the glutathione transferase reaction. The unprecedented stabilization of the supernucleophilic cob(I)alamin species under aerobic conditions by the intrinsic thiol oxidase activity of CblC was exploited for the chemoenzymatic synthesis of organocobalamin derivatives under mild conditions.

Derivatives of cobalamin, a complex organometallic cofactor, 1 support the activity of two mammalian enzymes . Unable to synthesize this essential cofactor, mammals use an elaborate pathway for converting dietary cobalamin to its active cofactor 2-4 forms and for delivering them to the two client enzymes . Functional B12 deficiency results from genetic defects in the cobalamin trafficking pathway and lead to systemic metabolic 5 defects . An early step in the B12 trafficking pathway involves 6 CblC, a versatile protein that exhibits glutathione transferase , 7 8 reductive decyanase and aquocobalamin (OH2Cbl) reductase activities and converts incoming cobalamin derivatives to an intermediate that can be partitioned into methylcobalamin (MeCbl) and 5´-deoxyadenosylcobalamin (AdoCbl) synthesis to support cellular needs. The glutathione transferase activity in2 volves an SN attack on the alkyl group of alkylcobalamins (RCbl) by the thiolate of glutathione (GSH) and leads to the heterolytic cleavage of the cobalt-carbon bond and formation of cob(I)alamin and the corresponding alkylthioether product (eq. 1). The decyanase activity requires a reductant in addition to cyanocobalamin (CNCbl), and leads to homolytic cleavage of the cobalt-carbon bond and formation of cob(II)alamin and cyanide (eq. 2). CblC can utilize reduced fla7,9 8 vins or GSH as an electron source. +

R-Cbl + GSH → GS-R + H + Cob(I)alamin -



+

[1]

CNCbl + GSH → CN + GS + H + Cob(II)alamin

[2]

Both the nucleophilic cob(I)alamin and paramagnetic cob(II)alamin derivatives are susceptible to oxidation and, with human CblC, convert rapidly to OH2Cbl under aerobic condi6,7 tions . In contrast, the C. elegans CblC (ceCblC) stabilizes cob(II)alamin formed during GSH-dependent decyanation of 8 CNCbl . The mechanism of stabilization involves efficient reduction of OH2Cbl by GSH and leads to formation of oxidized glutathione (GSSG). In this study, we report the remarkable aerobic stabilization of cob(I)alamin formed by ceCblC in the glutathione transferase reaction, which we have exploited for chemoenzymatic synthesis of organocobalamins under mild conditions. Recombinant full-length ceCblC was purified as described 8 previously and the thermodynamic parameters and stoichiometry (n ≈ 1) for binding of AdoCbl (KD = 13 ± 3 nM) and MeCbl (KD = 8 ± 1 nM) were determined by isothermal titration calorimetry (Table S1, Fig. S1). Dealkylation of AdoCbl was observed in the presence of GSH (5 mM) but not other thiols that were tested (5 mM each of dithiothreitol, β-mercaptoethanol, L-cysteine or Lhomocysteine). Strikingly, despite the reaction being conducted under aerobic conditions, formation of cob(I)alamin, characterized by an absorption maximum at 390 nm, was observed with isosbestic points at 354, 415 and 534 nm (Fig. 1A). In contrast, the two–electron oxidation product OH2Cbl, is observed during 6 aerobic dealkylation with human CblC . Upon prolonged incubation, cob(I)alamin was oxidized to cob(II)alamin as evidenced by an increase in absorption at 472 nm with isosbestic points at 358 and 413 nm (Fig. 1B). Further oxidation, i.e. conversion of cob(II)alamin to OH2Cbl was negligible over the next 2 h. At lower GSH concentrations (