Probing the mechanism and genomic context of CobT, the lower ligand activating enzyme in vitamin B12 biosynthesis

Abstract

Vitamin B12 is a cobamide cofactor required by many organisms including humans for catalysis of various reactions. It is a structurally complex small molecule that consists of a tetrapyrrolic corrin ring, an upper ligand and a lower ligand, 5,6-dimethylbenzimidazole (DMB) attached via a nucleotide loop. NaMN: DMB phosphoribosyl transferase CobT is the enzyme that activates the lower ligand prior to its attachment. In this study we aim to understand the regiospecificity and substrate preference of CobT homologs from obligate anaerobes that carry out de novo biosynthesis of lower ligand of vitamin B12. First based on gene neighbourhood of the encoding cobT gene, we classified the CobT homologs into three types, CobT1, CobT2 and CobT3. Then we reconstituted the activity of representative CobT1, CobT2 and CobT3 homologs and proved that only CobT1 homologs carry out a regiospecific activation of 5-OHBza. We studied the promiscuity in the substrate preference of the CobT homologs by characterizing their reactions with other naturally occurring lower ligands and proved that they are indeed catalytically promiscuous. Lastly, we studied the substrate preference of a representative CobT1 and CobT3 homolog proving that the most preferred substrate of CobT1 homolog is 5-OHBza whereas for the CobT3 homolog, it is DMB. In conclusion we establish that CobT1 homologs from obligate anaerobes differ vastly in their regiospecificity and substrate preference when compared to other CobT homologs. We also establish that an organism may possess multiple functional CobT enzymes which differ in activity. We also conclude that regardless of gene neighbourhood, CobT enzymes show promiscuity in their activity and can activate a variety of lower ligands.