Regiospecific formation of cobamide isomers is directed by CobT

Terence S. Crofts, Amrita B. Hazra, Jennifer LA Tran, Olga M. Sokolovskaya, Vadim Osadchiy, Omer Ad, Jeffrey Pelton, Stefan Bauer, Michiko E. Taga*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

Abstract

Cobamides, which include vitamin B12 (cobalamin), are a class of modified tetrapyrroles synthesized exclusively by prokaryotes that function as cofactors for diverse biological processes. Cobamides contain a centrally bound cobalt ion that coordinates to upper and lower axial ligands. The lower ligand is covalently linked to a phosphoribosyl moiety through an alpha-glycosidic bond formed by the CobT enzyme. CobT can catalyze the phosphoribosylation of a variety of substrates. We investigated the ability of CobT to act on either of two nitrogen atoms within a single, asymmetric benzimidazole substrate to form two isomeric riboside phosphate products. Reactions containing asymmetric benzimidazoles as substrates for homologues of CobT from different bacteria resulted in the production of distinct ratios of two isomeric products, with some CobT homologues favoring the production of a single isomer and others forming a mixture of products. These preferences were reflected in the production of cobamide isomers with lower ligands attached in different orientations, some of which are novel cobamides that have not been characterized previously. Two isomers of methoxybenzimidazolylcobamide were found to be unequal in their ability to support ethanolamine ammonia-lyase dependent growth in Salmonella enterica, suggesting that CobT's regiospeci ficity could be biologically important. We also observed differences in pKa, which can influence the reactivity of the cofactor and could contribute to these distinct biological activities. Relaxed regiospecificity was achieved by introducing a single point mutation in an active site residue of CobT. These new cobamide isomers could be used to probe the mechanisms of cobamide-dependent enzymes. (Graph Presented).

Original languageEnglish (US)
Pages (from-to)7805-7815
Number of pages11
JournalBiochemistry
Volume53
Issue number49
DOIs
StatePublished - Dec 16 2014

ASJC Scopus subject areas

  • Biochemistry

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