THE ROLE OF THE SALVAGE PATHWAY IN NUCLEOTIDE SUGAR BIOSYNTHESIS: IDENTIFICATION OF SUGAR KINASES AND NDP-SUGAR PYROPHOSPHORYLASES by TING YANG (Under the Direction of Maor Bar-Peled) ABSTRACT The synthesis of polysaccharides, glycoproteins, glycolipids, glycosylated secondary metabolites and hormones requires a large number of glycosyltransferases and a constant supply of nucleotide sugars. In plants, photosynthesis and the NDP-sugar inter-conversion pathway are the major entry points to form NDP-sugars. In addition to these pathways is the salvage pathway, a less understood metabolism that provides the flux of NDP-sugars. This latter pathway involves the hydrolysis of glycans to free sugars, sugar transport, sugar phosphorylation and nucleotidylation. The balance between glycan synthesis and recycling as well as its regulation at various plant developmental stages remains elusive as many of the molecular components are unknown. To understand how the salvage pathway contributes to the sugar flux and cell wall biosynthesis, my research focused on the functional identification of salvage pathway sugar kinases and NDP-sugar pyrophosphorylases. This research led to the first identification and enzymatic characterization of galacturonic acid kinase (GalA kinase), galactokinase (GalK), a broad UDP-sugar pyrophosphorylase (sloppy), two promiscuous UDP-GlcNAc pyrophosphorylases (GlcNAc-1-P uridylyltransferases), as well as UDP-sugar pyrophosphorylase paralogs from Trypanosoma cruzi and Leishmania major. To evaluate the salvage pathway in plant biology, we further investigated a sugar kinase mutant: galacturonic acid kinase mutant (galak) and determined if and how galak KO mutant affects the synthesis of glycans in Arabidopsis. Feeding galacturonic acid to the seedlings exhibited a 40-fold accumulation of free GalA in galak mutant, while the wild type (WT) plant readily metabolizes the fed-sugar. These findings suggest that in vivo, the GalAK gene product functions to salvage GalA in Arabidopsis. Interestingly, the galak mutant showed no visible morphological phenotype compared to WT, and the cell wall sugar composition was not affected in the mutant. Immunohistochemical analysis of galak indicated no glycan structural changes in galak. The information gained indicates that the gene encoding GalAK is required for proper recycling of GalA in plant cell. However, knocking out GalAK is not detrimental for plant cell wall synthesis, plant growth and development. INDEX WORDS: Nucleotide sugar, Salvage pathway, Sugar kinase, Nucleotide sugar pyrophosphorylase, Galacturonic acid kinase, Galactokinase, GlcNAc-1-P uridylyltransferase, UDP-GlcNAc pyrophosphorylase, Sloppy, Arabidopsis thaliana THE ROLE OF THE SALVAGE PATHWAY IN NUCLEOTIDE SUGAR BIOSYNTHESIS: IDENTIFICATION OF SUGAR KINASES AND NDP-SUGAR PYROPHOSPHORYLASES by TING YANG B.S., Jilin University, China, 2006 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 2011 © 2011 TING YANG All Rights Reserved THE ROLE OF THE SALVAGE PATHWAY IN NUCLEOTIDE SUGAR BIOSYNTHESIS: IDENTIFICATION OF SUGAR KINASES AND NDP-SUGAR PYROPHOSPHORYLASES by TING YANG Major Professor: Maor Bar-Peled Committee: Kelley Moremen Michael Tiemeyer Zachary Wood Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2011 DEDICATION I dedicate this work to my parents, for their love and encouragement; and to my husband, Tianhao He, for his caring support in my life. iv ACKNOWLEDGEMENTS I am deeply thankful to my major professor, Dr. Maor Bar-Peled, for his endless guidance and enthusiasm motivated me to the research. I would like to give my many thanks to all my committee members: Dr. Michael Tiemeyer, Dr. Kelley Moremen and Dr. Zachary Wood, for their support and valuable advice in my research. I am grateful for the help I received from Dr. John Glushka, for his guidance and assistance with the NMR experiments, I have learned much from him; I would also like to acknowledge Dr. Utku Avci, for his help with the immunohistochemistry assays; Dr. Yanbin Yin, for his help with the phylogeny analysis; Dr. Siva Kuma, for his help with the glycome profiling; I would also like to thank Dr. Malcolm O‟Neill for his assistance with the glycan analysis. In addition, I would like to thank all the present and former lab members who have shared their ideas with me, especially Dr. Xiaogang Gu, Dr. Yingnan Jiang and James Amor Smith, for their encouragement and great help in the research. I would like to show my gratitude to many undergraduate students who have worked with me: Andy Martin, Sung G Lee, Lindsey Gebhart and Ben Mullenbach; and to my best friends. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 PART I: NUCLEOTIDE SUGAR BIOSYNTHETIC SALVAGE PATHWAY ....1 PART II: THE EFFECTS OF MUTANTS IN THE SALVAGE PATHWAY ON PLANT DEVELOPMENT ....................................................................................31 PART III: COMPARATIVE ANALYSIS OF PROTEIN DOMAINS OF SUGAR KINASES AND NUCLEOTIDE SUGAR PYROPHOSPHORYLASES IN PLANTS ................................................................................................................36 PART IV: THESIS OVERVIEW ..........................................................................54 2 IDENTIFICATION OF GALACTURONIC ACID-1-PHOSPHATE KINASE, A NEW MEMBER OF THE GHMP KINASE SUPERFAMILY IN PLANTS, AND COMPARISON WITH GALACTOSE-1-PHOSPHATE KINASE ......................55 ABSTRACT ...........................................................................................................56 INTRODUCTION .................................................................................................57 EXPERIMENTAL PROCEDURES ......................................................................58 RESULTS ..............................................................................................................66 DISCUSSION ........................................................................................................91 vi 3 IDENTIFICATION OF A NOVEL UDP-SUGAR PYROPHOSPHORYLASE WITH A BROAD SUBSTRATE SPECIFICITY IN TRYPANOSOMA CRUZI ..............96 ABSTRACT ...........................................................................................................97 INTRODUCTION .................................................................................................98 EXPERIMENTAL PROCEDURES ....................................................................100 RESULTS ............................................................................................................105 DISCUSSION ......................................................................................................127 4 IDENTIFICATION AND CHARACTERIZATION OF A STRICT AND OF A PROMISCUOUS N-ACETYLGLUCOSAMINE-1-P URIDYLYLTRANSFERASES IN ARABIDOPSIS ............................................132 ABSTRACT .........................................................................................................133 INTRODUCTION ...............................................................................................134 EXPERIMENTAL PROCEDURES ....................................................................138 RESULTS ............................................................................................................143 DISCUSSION ......................................................................................................169 5 THE ROLE OF GALAK IN GLYCAN METABOLISM .........................................173 ABSTRACT .........................................................................................................174 INTRODUCTION ...............................................................................................175 EXPERIMENTAL PROCEDURES ....................................................................176 RESULTS ............................................................................................................184 DISCUSSION ......................................................................................................202 6 CONCLUSIONS........................................................................................................205 REFERENCES ............................................................................................................................207 vii APPENDICES A SUPPLEMENTAL FIGURES AND TABLES .........................................................232 B SUMMARY OF PRIMERS, ANTIBODIES, CLONING CONSTRUCTS AND TRANSGENIC PLANTS .........................................................................................260 viii CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW PART I: NUCLEOTIDE SUGAR BIOSYNTHETIC SALVAGE PATHWAY Introduction - Multiple routes for the synthesis of NDP-sugars Nucleotide sugars (Fig. 1.1, abbreviated NDP-sugars) are the substrates for the synthesis of polysaccharides, glycoproteins, glycolipids, glycoside-linked secondary metabolites and hormones. The biosynthesis of nucleotide sugars in plants is complicated and can be achieved via several pathways (Fig. 1.2): the inter-conversion pathway converts pre-existing NDP-sugars to other NDP-sugars (Reiter and Vanzin, 2001; Mohnen, 2002; Bar-Peled