Methylation reactions in the anaerobic biosynthesis of the lower ligand of vitamin B12

Abstract

Vitamin B12 belongs to the cobamides family of cofactors required by several organisms including bacteria, archaea, protists, algae, and animals. Cobamides act as cofactors for methyl transfer and radical-based reaction in a wide range of biological processes. However, the biosynthesis of B12 and all other cobamides is limited to a subset of bacteria and archaea. The biosynthesis of cobamides involves a modular pathway in which the structural components - the tetrapyrrolic cobalt ion containing corrin ring, the upper ligand, and the lower ligand - are synthesized independently and assembled together in the later steps in the pathway. In B12, the lower ligand is 5,6-dimethylbenzimidazole (DMB), however other cobamides contain a variety of lower ligands derived from benzimidazoles, purines, and phenols. The aim of my thesis was set to mechanistically investigating the recently discovered DMB biosynthesis bza operon in anaerobic bacteria. The first biosynthesis intermediate has been shown to be produced via a radical-SAM enzyme BzaAB/BzaF, and the remaining enzymes CobT, and BzaC, BzaD, and BzaE were to be characterized. The next gene cobT was predicted to encode a phosphoribosyltransferase that activates a wide range of lower ligands prior to cobamide assembly, and the genes bzaCDE were predicted to encode methyltransferases that yield distinctly methylated benzimidazole intermediates with DMB as the final product. To begin, we characterize the bza operon CobT by examining differences in its enzyme activity and substrate preferences in comparison to other CobT homologs from different gene neighborhoods. Next, our in-depth biochemical analyses with CobT and BzaC from the anaerobe Moorella thermoacetica reveal that the activation of benzimidazole precedes the methylation steps, shedding light on previously unexplained observations of the highly regiospecific lower ligand attachment in anaerobic B12 biosynthesis. Further, we find a novel domain of unknown function (DUF) 2284 present in some BzaC homologs and our primary biochemical studies establish the DUF2284 as an iron-sulphur cluster binding domain. We also observe the patterns of co-occurrence of this domain with the putative methyltransferase genes bzaD and bzaE in a comparative genomics study which in turn reveals the diversity in composition of the bza operon across anaerobic bacteria. Finally, as a part of our bioinformatic investigations, we identify homologs of CobT in a handful of eukaryotes which typically do not produce B12. We have initiated the studies with enzyme characterization and comparative genomics that will pave way for understanding cobamide metabolism in communities with microbial eukaryotes which have recently garnered attention in the B12 field. In summary, the insights we have gained from the bioinformatic, biochemical, and mechanistic explorations of the bza operon enzymes will aid in improving the industrial production of Vitamin B12 and other cobamides, as well as provide insights for metagenomic efforts to predict cobamide production and exchange in microbial communities.