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Functional Assignments in the Enolase Superfamily: Investigations of Two Divergent Groups of D-Galacturonate Dehydratases and Galactarate Dehydratase-Iii

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Functional Assignments in the Enolase Superfamily: Investigations of Two Divergent Groups of D-Galacturonate Dehydratases and Galactarate Dehydratase-Iii FUNCTIONAL ASSIGNMENTS IN THE ENOLASE SUPERFAMILY: INVESTIGATIONS OF TWO DIVERGENT GROUPS OF D-GALACTURONATE DEHYDRATASES AND GALACTARATE DEHYDRATASE-III BY FIONA PATRICIA GRONINGER-POE i 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 A. Gerlt, Chair Professor John E. Cronan, Jr. Associate Professor Rutilo Fratti Associate Professor Raven H. Huang ABSTRACT More than a decade after the genomic age, full genome sequencing is cost-effective and fast, allowing for the deposit of an ever increasing number of DNA sequences. New fields have arisen from this availability of genomic information, and the way we think about biochemistry and enzymology has been transformed. Unfortunately, there is no robust method for accurately determining the functions of enzymes encoded by these sequences that matches the speed in which genomes are deposited into public databases. Functional assignment of enzymes remains of utmost importance in understanding microbial metabolism and has applications in agriculture by examining bacterial plant pathogen metabolism and additionally in human health by providing metabolic context to the human gut microbiome. To aid in the functional identification of proteins, enzymes can be grouped into superfamilies which share common structural motifs as well as mechanistic features. To this end, the enolase superfamily is an excellent model system for functional assignment because more than half of the members still lack functional identification. Structurally, these enzymes contain substrate specificity residues in the N-terminal capping domain and catalytic residues in the C-terminal barrel domain. Mechanistically, each enzyme catalyzes the abstraction of a proton alpha to a carboxylate on the substrate before proceeding to dehydration, epimerization, deamination, racemization or cycloisomerization. There have been enough studies of this superfamily to provide valuable insight into the types of reactions performed based on catalytic residues and substrate specificity residues, laying the groundwork for functional characterization of unknown members. ii In this thesis, I address the problem of functional assignment by utilizing computational, structural, biochemical, and microbiological techniques to assign previously undiscovered functions to three groups of enzymes in the enolase superfamily that were previously unknown: the Microscilla group of D-galacturonate dehydratases, the Geobacillus group of D- galacturonate dehydratases, and galactarate dehydratase-III from Agrobacterium tumefaciens strain C58. Although the two groups of D-galacturonate dehydratases produce an identical product, they act through different mechanisms and have different structural elements. The functional assignments of these enzymes contribute to our understanding of the potential mechanisms and functions possible in this superfamily. The Microscilla group of D-galacturonate dehydratases (GalurDs) from Microscilla species PRE-1, Streptomyces coelicolor A3(2), Saccarophagus degradans 2-40, Pseudoalteromonas atlantica T6c and others utilize D-galacturonate to produce 5-keto-4-deoxygalacturonate in dehydration reaction consistent with known acid-sugar dehydratases in the enolase superfamily. These enzymes house a KxK motif on the second beta strand and a H/D dyad at the seventh and sixth beta strand in the barrel domain. These enzymes are found in organisms that degrade agar. In Microscilla species PRE-1 the GalurD gene is encoded on a plasmid along with other genes involved in agar degradation implying that GalurD could be contributing to the metabolism of agar. The Geobacillus group of D-galacturonate dehydratases from Geobacillus and Paenibacillus are structurally divergent from the abovementioned Microscilla group of GalurDs; unlike other acid- sugar dehydratases in the enolase superfamily, the Geobacillus GalurD contains an unusual iii second magnesium ion near the fourth beta strand of the barrel. Upon investigation, we believe this second magnesium is not catalytic and is rather an artifact of crystallization. Although these enzymes perform the same reaction as GalurDs from Microscilla, the Geobacillus group has a different mechanism and different sequence and is thus a separate group of GalurDs in the enolase superfamily. Galactarate dehydratase-III (GalrD-III) from Agrobacterium tumefaciens strain C58 is unlike previously reported galactarate dehydratases in the enolase superfamily in terms of sequence and substrate specificity: GalrD-III dehydrates galactarate with catalytic residues similar to galactonate dehydratases (galactonate dehydratases do not dehydrate galactarate, nor do galactarate dehydratases dehydrate galactonate) making this reaction unique in the enolase superfamily. Furthermore, GalrD-III dehydratases D-galacturonate by abstracting the proton located alpha to the aldehyde, producing a diketo product which undergoes a Benzil rearrangement to yield either 3-deoxy-D-xylo-hexarate or 3-deoxy-D-lyxo-hexarate (stereochemistry at C2 is uncertain), a compound not found in any known metabolic pathway. This is a novel mechanistic step and product in the enolase superfamily which prompts us to reexamine the possible substrates that can be utilized by enolase superfamily members. In providing these functional assignments to these groups of enzymes, we are not only able to extend these functions to other enolase superfamily members sharing the appropriate structural characteristics, but we can also expand our screening libraries and methods to incorporate aldose sugars; these have been previously overlooked as a possible substrate for sugar dehydratases in iv this superfamily. Thus, this work contributes to the problem of functional assignment by illuminating additional mechanistic and functional possibilities in the enolase superfamily. v For my darling husband Michael vi ACKNOWLEDGEMENTS I would like to gratefully acknowledge my thesis advisor, Dr. John A. Gerlt, without whom none of this research would have been possible. You have provided me with the knowledge and persistence necessary to succeed in science, and I will always be grateful for your efforts. Thank you for inspiring me to continually improve myself by setting a standard that will stay with me for the rest of my career. Thank you to my committee for their time throughout my graduate school career. I appreciate all you have done to shape me into a better scientist. In particular, thank you to Dr. Rudy Fratti for insight and useful discussions throughout my graduate school career; your class my first year helped me to understand how biochemists think. Thank you to Dr. John Cronan for providing a wonderful example of how to balance bench work with advising. Thank you to Dr. Raven Huang for always asking challenging questions. I hope these few words will suffice to express my immense appreciation and admiration. I would also like to thank my family especially my parents, Tim and Sue Mills-Groninger, whom were my first teachers and my inspiration for starting a career in science. Thank you for helping me to find my passion and always supporting me. Dad, thank you for your unending love, encouragement, advice, and terrible jokes; you continually inspire me to be a better person than you, which is thankfully not that difficult. Mom, I miss you and wish you could have lived to see this dream become reality, if only to gloat that you knew this day would come. vii Thank you to all the wonderful co-workers I have had the honor of working alongside during my time at UIUC: Dr. Tobias J. Erb, Dr. John F. Rakus, Dr. Kui Chan, Dr. Tiit Lukk, Dr. B. McKay Wood, Dr. Benjamin P. Warlick, Dr. Xinshaui Zhang, Salehe Ghasempur, Jason Bouvier, Bijoy Desai, and Dan Wichelecki. A special thank you to Dr. Kou-San Ju for the many experimental suggestions and being an almost endless font of expertise in microbiology and to Dr. James Doroghazi for his lovely PERLs which made several of my of my figures possible. I would also like to thank my friends and colleagues at UIUC including Dr. Dan Frank for his expertise in science as well as in beer; Dr. Amy Glekas for sharing her experiences and empowering me to take hold of my dreams; Dr. Heidi Imker for her unending wealth of enzymatic and scientific knowledge, as well as life-changing organizational tips; Dr. Stacy Kelley for support throughout my first years at UIUC; Sheena Smith for her incredible friendship and allowing me to live vicariously through her world travels; the lunch bunch at the IGB including Joel Cioni, Courtney Cox, Michelle Goettege, Joel Melby, Katie Molohon, and Spencer Peck for sharing their experiences with me; and finally my knitting group, without whom I would have surely wasted many Saturday nights. Finally, thank you to my husband Michael for his unending love and to my darling baby Squiggums for allowing me be a scientist by day and his mommy by night. viii Table of Contents LIST OF FIGURES ................................................................................................................... xiv LIST OF TABLES .................................................................................................................... xvii CHAPTER 1: INTRODUCTION ................................................................................................ 1 1.1. Genomic enzymology in the post-genomic era
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