FUNCTIONAL AND PHYSIOLOGICAL DISCOVERY IN THE MANNONATE DEHYDRATASE SUBGROUP OF THE ENOLASE SUPERFAMILY BY DANIEL JOSEPH WICHELECKI DISSERTATION Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2014 Urbana, Illinois Doctoral Committee: Professor John Gerlt, Chair Professor John Cronan Professor Scott Silverman Professor Wilfred van der Donk ABSTRACT In the current post-genomic world, the exponential amassing of protein sequences is overwhelming the scientific community’s ability to experimentally assign each protein’s function. The use of automated, homology-based annotations has allowed a reprieve from this efflux of data, but has led to widespread misannotation and nonannotation in protein sequence databases. This dissertation details the functional and physiological characterization of the mannonate dehydratase subgroup (ManD) of the enolase superfamily (ENS). The outcome affirms the dangers of homology-based annotations while discovering novel metabolic pathways. Furthermore, the experimental verification of these pathways ( in vitro and in vivo ) has provided a platform to test the general strategies for improved functional and metabolic characterization being developed by the Enzyme Function Initiative (EFI). Prior to this study, one member of the ManD subgroup had been characterized and was shown to dehydrate D-mannonate to 2-keto-3-deoxy-D-gluconate. Forty-two additional members of the ManD, selected from across the sequence space of the subgroup, were screened for activity and kinetic constants were determined. The members of the once isofunctional subgroup were found to differ in both catalytic efficiency and substrate specificity: 1) high 3 4 -1 -1 efficiency (k cat /K M = 10 to 10 M s ) dehydration of D-mannonate, 2) low efficiency (k cat /K M = 10 1 to 10 2 M-1s-1) dehydration of D-mannonate and/or D-gluconate, and 3) no-activity with either D-mannonate or D-gluconate (or any other acid sugar tested). The novel D-gluconate activity in this subgroup was investigated, and the mechanism of its enzymatic action was discovered. Physiologically, D-mannonate dehydration is essential to D-glucuronate metabolism. The D-mannonate dehydratase, UxuA, is not a member of the ENS. No uxu A genes are found in the ii genome of organisms with high efficiency ManDs. Through in vitro characterization and in vivo verification, a high efficiency ManD was discovered in Caulobacter crescentus CB15 that fulfills the same physiological role as UxuA and is an example of convergent evolution. The genomes of organisms with low efficiency members of the ManD subgroup generally have the uxu A gene. Therefore, they likely fulfill a different physiological role than the high efficiency ManDs. Their in vitro characterization and in vivo functional verification lead to the discovery of a novel L-gulonate metabolic pathway in Chromohalobacter salexigens DMS3043 where L-gulonate is converted to D-mannonate by a dehydrogenase and a reductase. While the low efficiency ManD found in C. salexigens is not metabolically essential to this pathway, its presence lead to the discovery of the pathway. Similar methods in Salmonella enterica subsp. enterica serovar Enteritidis str. P125109 lead to the discovery a novel L-idonate pathway where L-idonate is converted to D-gluconate by two dehydrogenases and then dehydrated (the traditional pathway phosphorylates D-gluconate). This pathway directly involves a low efficiency GlcD that is a member of the ManD subgroup and raises interesting questions about the physiological role of low efficiency enzymes and redundant pathways. As a whole, this dissertation displays how function diverges as sequence diverges while laying bare the dangers of annotation via homology; concurrently, it demonstrates how the continually advancing assignment strategies of the EFI can be used to discover new enzymatic functions and metabolic pathways. iii ACKNOWLEDGEMENTS I would like to thank my thesis committee (Dr. John Cronan, Dr. Scott Silverman, and Dr. Wilfred van der Donk) for taking the time to check my progress, give advice, and ask challenging questions; the Cellular and Molecular Biology Training Grant for financial support; Dr. John Rakus for discovering the first D-mannonate dehydratase in the enolase superfamily; former and present members of the Gerlt lab (Jason Bouvier, Bijoy Desai, Salehe Ghasempur, Fiona Poe, Dr. Tiit Lukk, Dr. Ayano Sakai*, Dr. Xinshuai Zhang, and Dr. Ben Warlick); and the former and present members of the Microbiology core of the Enzyme Function Initiative (EFI) (Dr. Kyuil Cho, Dr. Brad Evans, Dr. Amy Jones*, Dr. Ritesh Kumar, Dr. Indu Rupassara, Dr. Jose Solbiati, Dr. Brian San Francisco, and Dr. Bryant McKay Wood). I would like to thank the Protein Production Core for the monumental amount of work they have contributed to my thesis research, including protein purification, protein crystallization, and ThermoFluor screening (Dr. Steve Almo, Dr. Nawar Al-Obaidi, Dr. Alexander Federov, Dr. Brandan Hillerich, Dr. Yuriy Patskovsky, Dr. Rafael Toro, and Dr. Matt Vetting). My best to the many talented undergraduate researchers I have mentored over the years (Bryan Balthazar, Anthony Chau, Dylan Graff, and Jean Vendiola); they took years off my “to graduation” time. I thank everyone for putting up with my automaton-like work persona, and for getting to know me more outside of work (most prominently Jason Bouvier, Anthony Chau, Dr. Ayano Sakai, Dr. Brian San Francisco, and Dr. Ben Warlick). I would also like to specifically thank Dr. Ayano Sakai, who trained me in almost every experimental technique I learned in this 5 year experience. Her patience, knowledge, and friendship will never be forgotten. Above all, I would like to thank my advisor and mentor, Dr. iv John Gerlt for his support and guidance through my PhD experience. Who, while beset by the immense time burden of directing the EFI, always found time to discuss my ManDs when I needed advice. I never imagined how much I would grow as a scientist under his influence. I thank him most for being honest in criticism and free with praise. Most importantly, I would like to thank my fiancée, Dr. Katie Whalen, whose companionship, patience, love, and writing skill has carried me safely over the many hurdles I’ve encountered in the PhD process. Naturally, I thank my loving family members for all of there support through the years: Mom, Dad, Jana, Steven, and Cassidy. v TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... xi LIST OF TABLES ..................................................................................................................... xiv LIST OF SCHEMES ...................................................................................................................xv CHAPTER 1: INTRODUCTION .................................................................................................1 1.1 Focus of Study ..........................................................................................................................1 1.2. Dilemma of the Post-Genomic Era ...........................................................................................1 1.2.1. Miss- and Non-annotation in Protein Sequence Databases ....................................... 3 1.3. The Enzyme Function Initiative................................................................................................4 1.3.1. Strategy of the EFI ....................................................................................................4 1.4. The Enolase Superfamily ..........................................................................................................6 1.4.1. The Mannonate Dehydratase Subgroup .................................................................10 1.4.2. Fuconate Dehydratases in the Mandelate Racemase Subgroup ............................12 vi CHAPTER 2: DISCOVERY OF FUNCTION IN THE ENOLASE SUPERFAMILY; D-MANNONATE AND D-GLUCONATE DEHYDRATASES IN THE D-MANNONATE DEHYDRATASE SUBGROUP .....................................13 2.1. Introduction .............................................................................................................................14 2.2. Materials and methods ............................................................................................................17 2.3. Results and discussion ............................................................................................................23 2.4. Conclusions .............................................................................................................................35 CHAPTER 3: IDENTIFICATION OF THE IN VIVO FUNCTION OF THE HIGH EFFICIENCY D-MANNONATE DEHYDRATASE IN CAULOBACTER CRESCENTUS NA1000 FROM THE ENOLASE SUPERFAMILY ..............37 3.1. Introduction .............................................................................................................................37 3.2. Materials and methods ............................................................................................................41 3.3. Results and discussion ............................................................................................................45 3.4. Conclusions .............................................................................................................................49