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Genetic and Biochemical Origins of Diversity in Cobamides: Nature's Genetic and Biochemical Origins of Diversity in Cobamides: Nature’s Most Beautiful Cofactors By Terence Spencer Crofts A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate Division of the University of California, Berkeley Committee in charge: Professor Michiko E. Taga, Chair Professor Michelle Chang Professor Krishna Niyogi Professor Patricia C. Zambryski Fall 2013 Abstract Genetic and Biochemical Origins of Diversity in Cobamides: Nature’s Most Beautiful Cofactors by Terence Spencer Crofts Doctor of Philosophy in Microbiology University of California, Berkeley Professor Michiko E. Taga, Chair 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). These 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). The 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. The 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 1 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. Across 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. 2 Table of Contents Chapter 1 ....................................................................................................................................... 1 1.1 Introduction ...................................................................................................................... 1 1.2 Brief history of cobalamin................................................................................................ 1 1.3 Cobalamin-dependent reactions ....................................................................................... 4 1.4 The biosynthesis of cobalamin ......................................................................................... 6 1.5 Cobalamin is only one representative of a large family of cobamide cofactors .............. 7 1.6 Biochemical and genetic origins of cobamide diversity ................................................ 10 1.7 Significance of cobamide variation ................................................................................ 20 1.8 Conclusion ...................................................................................................................... 22 Chapter 2 ..................................................................................................................................... 23 2.1 Summary ........................................................................................................................ 23 2.2 Introduction .................................................................................................................... 23 2.3 Results ............................................................................................................................ 27 2.4 Discussion ...................................................................................................................... 35 2.5 Significance .................................................................................................................... 37 2.6 In vitro studies of CobT substrate specificity................................................................. 38 2.7 Materials and Methods ................................................................................................... 38 Chapter 3 ..................................................................................................................................... 43 3.1 Summary ........................................................................................................................ 43 3.2 Introduction .................................................................................................................... 43 3.3 Results ............................................................................................................................ 46 3.4 Discussion ...................................................................................................................... 58 3.5 Materials and Methods ................................................................................................... 62 Chapter 4 ..................................................................................................................................... 66 4.1 Summary ........................................................................................................................ 66 4.2 Introduction .................................................................................................................... 66 4.3 Results ............................................................................................................................ 68 4.4 Discussion ...................................................................................................................... 76 4.5 Materials and Methods ................................................................................................... 78 Chapter 5 ..................................................................................................................................... 81 5.1 Dissertation results in context ........................................................................................ 81 5.2 Unanswered questions in cobamide research ................................................................. 83 References .................................................................................................................................... 85 i Acknowledgements During the past several weeks of writing this dissertation, I have been reminded of all the help and effort others have given me. Of these I need to first thank Michi Taga for being a great mentor and advisor. When I joined the lab, Michi gave me a chance to figure out what I wanted to do and she has been very supportive ever since. She taught me how to be a careful experimenter and a better writer. I am also very grateful for the freedom she gave me by allowing me to choose what questions I wanted to answer while also always providing guidance. I also want to thank the other members of my qualifying exam and dissertation committees: Bob Buchanan, David Zusman, Pat Zambryski, and Michelle Chang. I received a lot of valuable feedback from meetings with this group of professors and their suggestions (e.g. Gibson cloning) improved the pace and quality of my research. I also want to acknowledge my undergraduate advisor, David Kranz, for first letting me join his lab and showing me how fun research can be. My fellow Taga lab researchers have also been great companions and sources of valuable advice, both technical and philosophical. I have been very lucky
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