Executive Summary : | Vitamins are essential micronutrients required for cellular metabolism across all domains of life. Also known as coenzymes/ cofactors, vitamins assist enzymes in catalyzing chemically challenging reactions such as isomerization, oxidation-reduction and radical-based rearrangements under biological conditions. Cobalamin (vitamin B12) is one such important micronutrient essential for humans for methionine biosynthesis and for the breakdown of methylmalonyl CoA, a toxic byproduct of metabolism. Cobalamin is just one member of the large cobamide family of cofactors, which are a set of naturally occurring cofactor analogues sharing a common tetrapyrrolic scaffold with a central cobalt ion coordinated to upper and lower ligands. The difference between cobamide family members arises due to the structure of the lower ligand, and organisms, including humans, often have a strict preference for one cobamide over another based on the lower ligand. Among all vitamins, cobalamin and the cobamide analogues stand out uniquely as they are synthesized only by microorganisms – a subset of bacteria and archaea - even though several organisms in the natural world require them. Cobamide synthesis requires more than 30 enzymes and is a metabolically expensive endeavor, thus the sharing of these molecules often forms the basis of microbe-microbe and host-microbe interactions. For example, cobamide sharing plays a central role in sustaining important microbial consortia such as the human gut microbiota, methanogenesis, acetogenesis and bioremediation communities. Interestingly, even though the enzymes involved in the cobamide biosynthesis pathway have been under examination for a few decades, the discovery of genes for lower ligand biosynthesis and attachment occurred only in the last decade and the study of their mechanisms is yet nascent. Also, no cobamide other than cyano- and methyl- forms of cobalamin are commercially available today, and hence studies of microbe-microbe and host-microbe interactions involving cobamides are very limited. The goal of this project is to understand the molecular mechanism of lower ligand choice in cobamide biosynthesis, and tailor it for facilitating the efficient production of cobalamin and a variety of cobamides. Specifically, using bioinformatics, molecular biology, mechanistic enzymology, and microbial genetics, we wish to (i) analyze several homologs of the lower-ligand activating and attachment enzymes, CobT and CobS, respectively, to predict the residues determining the molecular basis of substrate choice (ii) engineer specific CobT and CobS enzymes using mutational analysis to activate specific lower ligands and (iii) test these engineered enzymes by heterologous expression in Escherichia coli and in Propionibacterium freudenreichii, the commercial cobalamin producer strain, with a goal of producing a variety of cobamides at a laboratory scale and ultimately at commercial scales, respectively. |