Investigating the regioselective attachment of the lower ligand in Vitamin B12 biosynthesis

Abstract

Vitamin B12 (cobalamin), a member of the cobamide family of cofactors, is important for many organisms including humans, and facilitates diverse metabolic processes such as the biosynthesis and catabolism of amino acids, fatty acid metabolism, modification of tRNA, reductive dehalogenation and acetogenesis. The structure of Vitamin B12 and other cobamides consists of a tetrapyrrolic corrin ring, with a central cobalt ion, and an upper and a lower ligand. The upper ligand in cobamides is involved in the activity of the cofactor – catalyzing methyl transfer reactions and radical-based rearrangement reactions. Different cobamide cofactors vary mainly in the composition of their lower ligand which is covalently attached to the corrin ring via a nucleotide loop and may coordinate to the cobalt ion. 5, 6-dimethylbenzimidazole (DMB) is the lower ligand of Vitamin B12. The biosynthesis of cobamides can occur via an aerobic or an anaerobic route and typically consists of three main steps – the synthesis of the corrin ring, synthesis of the lower ligand and finally, their attachment to form the complete cobamide. The last step is initiated by CobT, an enzyme that couples the lower ligand to a ribophosphate derivative. This is further coupled to the nucleotide loop of the corrin ring with the enzymes CobU, CobS and CobC. The anaerobic biosynthesis of DMB was studied in the anaerobic B12 producer Eubacterium limosum and found to derive from the purine biosynthesis pathway intermediate 5'-phosphoribosyl-5-aminoimidazole (AIR). Despite being a symmetric molecule, labelling studies in the E. limosum show that DMB is attached selectively via one nitrogen. This suggests that the mechanism of attachment of the lower ligand is regioselective in nature. In my thesis project, I focus on investigating the mechanism by which regioselectivity is conferred in the attachment of the lower ligand of B12. In E. limosum, enzymes of the bza operon sequentially convert AIR to DMB via three asymmetric precursors, 5-hydroxybenzimidazole, 5-methoxybenzimidazole and 5-methoxy, 6-methylbenzimidazole. The enzyme CobT, also a part of the bza operon in vi E. limosum, attaches the lower ligand to the corrin nucleotide loop. To explain the selective attachment of one nitrogen in DMB, we hypothesized that either an asymmetric precursor of DMB may be the substrate for CobT, or that enzyme complex of the bza operon interacts with CobT, causing anisotropy in DMB biosynthesis that leads to the regioselectivity. In this study, we show that the E. limosum CobT is regioselective for certain asymmetric benzimidazole ligands, and compared it with the activity of Escherichia coli CobT. Further, using bioinformatic analysis and the crystal structure of the Salmonella enterica CobT , we have identified key residues that are involved in the regioselective attachment of lower ligands and demonstrated that these residues affects the selectivity displayed by CobT. We also show that factors such as pH or enzyme-enzyme interactions have little or no effect on the regioselectivity of CobT. Our study facilitates the understanding of the molecular basis of attachment of the lower ligand in cobamide biosynthesis. This will contribute to research on mechanisms by which microbes create cobamide diversity by varying the lower ligand. Our analysis not only deepens the understanding of the mechanism of CobT, but also makes it amenable to protein engineering so as to improve the production of Vitamin B12 and other cobamide cofactors.