Genetic and Biochemical Origins of Diversity in Cobamides: Nature's Most Beautiful Cofactors

The cobamide class of small molecules includes the essential micronutrient cobalamin (Vitamin B12). Cobamides catalyze a variety of reactions involving one-carbon transfers and radical mediated molecular rearrangements. These reactions are vital parts of biochemical pathways found in both humans (one-carbon metabolism, fatty acid metabolism) and prokaryotes (methanogenesis, acetogenesis, fermentation and bioremediation)./nThese chemically difficult reactions are made possible by the complex structure of cobamides. Cobamides are tetrapyrroles related to heme, chlorophyll and Factor F430, but are unique in containing a cobalt atom that binds an upper and lower ligand. The upper ligand of cobamide cofactors is made up of a cobalt-carbon covalent bond and varies depending on the metabolic role of the cofactor. The lower ligand can vary widely and is largely responsible for the diversity of cobamides found in nature. Lower ligands can take the form of benzimidazoles (5,6-dimethylbenzimidazole in the case of cobalamin), purines (such as adenine), or phenolics (such as p-cresol)./nThe first chapter of my thesis will provide more background into the functions and structures of cobalamin and other cobamides. I will focus on the known diversity of cobamides in nature and how that diversity is the result of the metabolisms of cobamide producing organisms./nThe next three chapters detail a series of experiments that I have performed to expand our knowledge of how cobamide diversity is maintained. The first set of experiments (Chapter 2) describes how the CobT enzyme, the first enzyme in the lower ligand attachment pathway, is responsible for determining which lower ligands are incorporated into cobamides. I examined this at the organismal and genetic levels and collaborated on a companion project examining these mechanisms at the enzymatic level. I also demonstrated that altering this specificity can have deleterious effects on the host organism. In Chapter 3, building on the previous chapter, I and a collaborator in the laboratory (Amrita Hazra) conducted a series of experiments to show that some CobT enzymes also specify the orientation of the cobamide lower ligand. Organismal, genetic, and biochemical analyses showed that CobT enzymes and cobamide producing bacteria can synthesize pairs of products as structural isomers. These findings add a new potential source of structural variability in cobamides and reveal that CobT specificity extends to the orientation as well as the identity of the lower ligand. Finally, Chapter 4 describes a bioassay I developed to detect benzimidazole lower ligands in the environment and the results of its application to a variety of samples. I found that benzimidazoles are ubiquitous in the environment, a finding that has important implications for cobamide diversity in microbial communities./nAcross these experiments I have explored the means by which cobamide diversity is maintained in nature through the actions of CobT and the availability of lower ligand bases. The final chapter summarizes my findings and places them in the context of the field. This section also contains novel hypotheses generated by my results about the significance of cobamide diversity in nature as well as future experiments to test these hypotheses.

Advisor: Taga, Michiko E