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Biochemistry. 2014 Dec 16;53(49):7805-15. doi: 10.1021/bi501147d. Epub 2014 Dec 5.

Regiospecific formation of cobamide isomers is directed by CobT.

Author information

1
Department of Plant and Microbial Biology, ∥Department of Chemistry, ⊥QB3 Institute, and #Energy Biosciences Institute, University of California , Berkeley, United States.

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 regiospecificity 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.

PMID:
25412146
DOI:
10.1021/bi501147d
[Indexed for MEDLINE]

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