Biotin Synthesis in Escherichia Coli by Steven
Total Page：16
File Type：pdf, Size：1020Kb
BIOTIN SYNTHESIS IN ESCHERICHIA COLI BY STEVEN LIN DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology in the Graduate College of the University of Illinois at Urbana-Champaign, 2012 Urbana, Illinois Doctoral Committee: Professor John E. Cronan, Chair Professor James A. Imlay Professor William W. Metcalf Assistant Professor Carin Vanderpool ABSTRACT Biotin is an essential enzyme cofactor required by all three domains of life. It functions as a covalently-bound prosthetic group, which mediates the transport of CO2 in many vital metabolic carboxylation, decarboxylation and transcarboxylation reactions. Although biotin is essential, our knowledge of its biosynthesis remains fragmentary. Studies suggest that most of the carbon atoms of biotin are derived from pimelic acid, a seven carbon dicarboxylic acid. However, the mechanism whereby Escherichia coli assembles this pimelate intermediate was unclear. Genetic analyses identified only two genes of unknown functions, bioC and bioH, which are required for pimelate synthesis. BioC is annotated as an S-adenosyl-L-methionine (SAM) dependent methyltransferase, whereas BioH has shown carboxylesterase activity. The mechanism by which a methyltransferase and a carboxylesterase catalyze the synthesis of pimelate intermediate was very puzzling. In this Thesis, I describe my approaches to delineate E. coli biotin synthetic pathway and to elucidate the roles of BioC and BioH in pimelate synthesis. In Chapter 2, I unravel the synthesis of pimelate. I report in vivo and in vitro evidence that the pimelate intermediate is synthesized by a modified fatty acid synthetic pathway. The ω-carboxyl group of a malonyl-thioester precursor is methylated by BioC, as an initiation step in biotin synthesis. The shielding by a methyl ester moiety is required for recognition and chain elongation of this atypical substrate by the fatty acid synthetic enzymes. The malonyl-thioester methyl ester enters fatty acid synthesis as the primer and undergoes two reiterations of the fatty acid elongation cycle to give pimeloyl- acyl carrier protein (ACP) methyl ester. The methyl ester moiety is then cleaved by BioH to signal termination of elongation. The product pimeloyl-ACP then enters the second half of biotin synthetic pathway, and becomes the substrate of BioF reaction to begin biotin ring assembly. In Chapter 3, I demonstrate BioC methylation of malonyl-ACP, which is a key initiation reaction in E. coli biotin synthesis. I hypothesized that BioC catalyzes the transfer the methyl group from SAM to the ω-carboxyl group of malonyl-ACP, creating a ii methyl ester moiety. The methyl ester moiety is essential to allow processing of malonate, a C3 dicarboxylate, into pimelate, a C7 dicarboxylate by fatty acid synthetic enzymes. To demonstrate BioC activity experimentally, I cloned and purified Bacillus cereus BioC, which is the only amenable BioC homolog I found in several different bacterial species. By using radiolabeled SAM, I show that BioC specifically selects malonyl-ACP for methylation. Furthermore, this methylation activity is also susceptible to inhibition by molecules known to target SAM-dependent enzymes. In Chapter 4, I report a 2.0-Å resolution co-crystal structure of BioH in complex with its substrate, pimeloyl-ACP methyl ester. This structure was obtained in collaboration with the Satish Nair lab at University of Illinois at Urbana Champaign. BioC methylates the free carboxyl of a malonyl-thioester which replaces the usual acetyl- thioester primer. This atypical primer is transformed to pimeloyl-ACP methyl ester by two cycles of fatty acid synthesis. The question is what stops this C7-ACP from undergoing further elongation, to azelaryl (C9)-ACP methyl ester, a metabolically useless product. Although BioH readily cleaves this product in vitro, as shown in Chapter 2, the enzyme is nonspecific which made assignment of its physiological substrate problematical. The downstream enzyme BioF, which releases ACP as a byproduct, could theoretically also perform this “gatekeeping” function. We utilized the structure to demonstrate that BioH is the gatekeeper and its physiological substrate is pimeloyl-ACP methyl ester. Moreover, the binding interaction with ACP is important for BioH activity. In Chapter 5, I summarize my findings in E. coli biotin synthesis. I discuss my experimental approaches and technical troubleshooting that led to successful delineation of this pathway. I also describe the serendipitous discovery of pimeloyl-ACP methyl ester as a novel biotin intermediate. This intermediate led to the identifications of methyl ester moiety and ACP, which were the two missing puzzles to a complete understanding of E. coli biotin synthesis. Finally, I offer two future directions to investigate BioC structures, and to identify the 3-ketoacyl-ACP synthases involved in chain elongation of dicarboxylates. iii ACKNOWLEDGEMENTS When I was daydreaming out of boredom in an introductory Microbiology class in my sophomore year, getting a Ph.D. in Microbiology would be the last thing to cross my mind. I was generally interested in science and biology, but lacked the motivation or dedication to excel in my classes. After completing an undergraduate degree, I was lucky to find a research position in Academia Sinica, a government-funded research institute in Taiwan. Thanks to my mentors, Chun-Hung Lin and Tzann-Shun Hwang, I fell in love with biochemical research right away. They patiently taught me everything from scratch, believed in my potential, and encouraged me to pursue higher degrees. But my poor grades came back to haunt me. When I was overwhelmed by rejection letters, the Ohio State University offered me a second chance to the Master’s degree program. For this, I am forever grateful to Peng Wang, Venkat Gopalan and my thesis advisor, Donald Dean. I am very glad to come to the University of Illinois and join the Department of Microbiology, where I had the perfect combination of thesis advisor, research projects, committee members and colleagues. I thank my advisor, John E. Cronan, for allowing me to creatively explore my project and for providing guidance whenever I was lost. Not only have I vastly increased my repertory of research techniques, but I have also learned to think critically, execute efficiently and judge the outcome maturely. Of course, I could not have earned this degree without the generous help and honest advice from my committee members, and more importantly, from my colleagues in the Cronan lab. I want them to know that their support is absolutely vital to my research accomplishment. Finally, I want to dedicate this thesis to my family. I wish to thank my parents and sister for encouragement and for always believing in me. I want to thank my dear wife especially, for being loving and understanding through the ups and downs of my graduate study. She provided both physical and emotional nutrients to keep my research going strong. iv TABLE OF CONTENTS Chapter 1: Background and Significance ............................................................................1 The Discovery of Biotin...........................................................................................1 The Catalytic Role of Biotin ....................................................................................1 Biotin Synthesis .......................................................................................................2 7-Keto-8-Aminopelargonic Acid Synthase..................................................3 7,8-Diaminopelargonic Acid Synthase ........................................................4 Dethiobiotin Synthetase ...............................................................................5 Biotin Synthase ............................................................................................6 The Synthesis of Pimelate ........................................................................................8 BioC-BioH Pathway of Escherichia coli ...................................................10 BioI-BioW Pathway of Bacillus subtilis ....................................................12 Other Pathways of Pimelate Synthesis ......................................................14 The Aim and Scope of this Thesis .........................................................................16 Figures....................................................................................................................18 Chapter 2: Biotin Synthesis Proceeds by Hijacking the Fatty Acid Synthetic Pathway ..............................................................................................................................24 Introduction ............................................................................................................24 Materials and Methods ...........................................................................................25 Results ....................................................................................................................31 Discussions ............................................................................................................36 Tables .....................................................................................................................39 Figures....................................................................................................................44 Chapter 3: Methylation of Malonyl-Acyl-Carrier Protein by Bacillus cereus