HETEROLOGOUS EXPRESSION OF THE GAUT1:GAUT7 COMPLEX AND MECHANISMS OF HOMOGALACTURONAN BIOSYNTHESIS by ROBERT ALEXANDER AMOS (Under the Direction of Debra Mohnen) ABSTRACT Homogalacturonan (HG) is a pectic glycan in the plant cell wall that contributes to plant growth and development, cell wall structure and function, and interacts with other glycans and proteoglycans in the wall. HG is synthesized by the galacturonosyltransferase (GAUT) gene family. Two members of this family, GAUT1 and GAUT7, form a heteromeric enzyme complex in Arabidopsis thaliana. Progress in studying plant cell wall glycosyltransferases (GTs) has been limited by the difficulty of purifying these low abundance enzymes from native plant tissues and slow progress in developing viable heterologous expression systems. Here, we established a heterologous GAUT expression system in HEK293 cells and showed that co-expression of recombinant GAUT1 with GAUT7 results in the production of a soluble GAUT1:GAUT7 complex that catalyzes elongation of HG products in vitro. The reaction rates, progress curves, and product distributions exhibited major differences dependent upon small changes in the degree of polymerization (DP) of the oligosaccharide acceptor. GAUT1:GAUT7 displayed > 45- fold increased catalytic efficiency with DP11 acceptors relative to DP7 acceptors. Although GAUT1:GAUT7 synthesized high molecular weight polymeric HG (> 100 kDa) in a substrate concentration-dependent manner typical of distributive (non-processive) glycosyltransferases with DP11 acceptors, reactions primed with short-chain acceptors resulted in a bimodal product distribution of glycan products that has previously been reported as evidence for a processive model of GT elongation. As an alternative to the processive glycosyltransfer model, a two-phase distributive elongation model is proposed in which a slow phase, which includes the de novo initiation of HG and elongation of short-chain acceptors, is distinguished from a phase of rapid elongation of intermediate and long-chain acceptors. Upon reaching a critical chain length of DP11, GAUT1:GAUT7 elongates HG to high molecular weight products. Two-phase elongation is potentially a common feature of polysaccharide synthesis and may explain why many GTs yield product distributions that deviate from the results predicted for strictly processive or distributive mechanisms of synthesis. The high yield of GAUT1:GAUT7 expressed in HEK293 cells and the thorough biochemical and kinetic characterization described here provides a standard for future studies of polysaccharide biosynthesis from plant cell walls and other organisms. INDEX WORDS: plant cell wall, glycosyltransferase, protein complex, enzyme mechanism, processivity, homogalacturonan, pectin, pectic glycan, galacturonosyltransferase, polygalacturonide transferase HETEROLOGOUS EXPRESSION OF THE GAUT1:GAUT7 COMPLEX AND MECHANISMS OF HOMOGALACTURONAN BIOSYNTHESIS by ROBERT ALEXANDER AMOS B.S., University of Georgia, 2009 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2019 © 2019 Robert Alexander Amos All Rights Reserved HETEROLOGOUS EXPRESSION OF THE GAUT1:GAUT7 COMPLEX AND MECHANISMS OF HOMOGALACTURONAN BIOSYNTHESIS by ROBERT ALEXANDER AMOS Major Professor: Debra Mohnen Committee: Maor Bar-Peled David Garfinkel Ron Orlando Zachary Wood Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia May 2019 DEDICATION To Mom and Dad for endless love and support. iv ACKNOWLEDGEMENTS I would like to extend a special show of gratitude to my mentor, Dr. Debra Mohnen, for providing me with this opportunity and for persisting with me during the difficult trials of this project. Completion of this project was only possible with the assistance of many of my colleagues at the Complex Carbohydrate Research Center and the Department of Biochemistry and Molecular Biology. I would like to thank the members of my research committee: Dr. Zac Wood, Dr. David Garfinkel, Dr. Ron Orlando, and Dr. Maor Bar-Peled for the guidance necessary to direct this project to a successful conclusion, as well as Dr. Melani Atmodjo, Dr. Ajaya Biswal, Dr. Li Tan, and all members of the Mohnen lab. I also thank our collaborators Dr. Kelley Moremen, Dr. Jeong-Yeh Yang, Dr. Breeanna Urbanowicz, Dr. Sivakumar Pattathil, and everyone else who took time to provide me with your knowledge and good ideas, especially Dr. Malcolm O’Neill, Dr. Alan Darvill, Dr. Carl Bergmann, Dr. Michael Hahn, Dr. David Blum, Dr. Michael Adams, Dr. Lance Wells, Dr. Dusty Brown, Dustin Middleton, and DJ Bernsteel. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ....................................................................................................................... xi INTRODUCTION ...........................................................................................................................1 CHAPTER 1 LITERATURE REVIEW: GLYCOSYLTRANSFERASE ACTIVITIES OF HETEROLOGOUSLY-EXPRESSED PLANT CELL WALL MATRIX POLYSACCHARIDE BIOSYNTHETIC GENES AND THEIR SIGNIFICANCE FOR WALL POLYMER FUNCTION ..........................................................................6 Abstract ....................................................................................................................7 Introduction: Plant cell walls as quality factors in fruit and other plant-derived commercial products ................................................................................................8 Models of crosslinked plant cell wall polysaccharide networks ............................10 Strategies for identifying cell wall glycosyltransferase (CWGT) activities ..........13 Heterologous expression systems for plant cell wall GTs .....................................26 The biosynthesis of extended-chain backbone polymers .......................................30 Enzymatic rates and kinetic analyses .....................................................................41 Heterologous expression of complexes .................................................................44 Conclusion .............................................................................................................47 vi TABLES ................................................................................................................49 2 A TWO-PHASE MODEL FOR THE NON-PROCESSIVE BIOSYNTHESIS OF HOMOGALACTURONAN POLYSACCHARIDES BY THE GAUT1:GAUT7 COMPLEX ..................................................................................................................65 Abstract ..................................................................................................................66 Introduction ............................................................................................................67 Results ....................................................................................................................70 Discussion ............................................................................................................103 Experimental procedure .......................................................................................110 Supplementary table.............................................................................................118 CONCLUSIONS..........................................................................................................................119 REFERENCES ............................................................................................................................132 APPENDICES A ACTIVITY VARIATION OF GAUT1:GAUT7 PRODUCED IN HEK293 CELL CULTURES ....................................................................................................155 B ACTIVITY OF GAUT1:GAUT7 ACTIVE-SITE DXD MUTANTS .......................167 C PURIFICATION OF ACTIVE ENZYMES BY SIZE EXCLUSION CHROMATOGRAPHY TO REMOVE PROTEIN AGGREGATES ......................186 D STABILITY OF GAUT1:GAUT7 AND GAUT1 ENZYMES FOLLOWING NICKEL-AFFINITY PURIFICATION.....................................................................194 E EFFORTS TO DETERMINE THE SPECIFIC ACTIVITY OF GAUT1 RELATIVE TO GAUT1:GAUT7 ..................................................................................................201 F STOICHIOMETRY OF THE GAUT1:GAUT7 COMPLEX....................................207 vii G ELISA-BASED HG:GALAT ACTIVITY ASSAY ..................................................216 H COMPARISON OF METHODS FOR ASSAYING HG:GALAT ACTIVITY........224 viii LIST OF TABLES Page Table 1.1: Plant cell wall GTs of known function determined by heterologous expression .........49 Table 1.2: Chain length of plant cell wall polysaccharides synthesized in vivo and in vitro ........58 Table 1.3: Enzymatic rates and reaction kinetics of plant cell wall polysaccharides synthesized in vitro ............................................................................................................61 Table 1.4: Data from references used to calculate enzymatic rates in Table 1.3 ...........................64 Table 2.1: Results of Michaelis-Menten kinetics assays experiments of the Arabidopsis heterologously-expressed GAUT1:GAUT7