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Structure Characterization of a Peroxidase from The STRUCTURE CHARACTERIZATION OF A PEROXIDASE FROM THE WINDMILL PALM TREE TRACHYCARPUS FORTUNEI A DISSERTATION SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAWAI‘I AT MĀNOA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOSCIENCES AND BIOENGINEERING MAY 2015 By Margaret R. Baker Dissertation Committee: Qing X. Li, Chairperson Jon-Paul Bingham Dulal Borthakur Ho Leung Ng Monika Ward Keywords: palm tree, peroxidase, N-glycosylation, mass spectrometry © 2015, Margaret R. Baker ii DEDICATION I dedicate this work to my husband, Adam, a constant source of support and encouragement. I also dedicate it to my sister, Lydia and my nephews, Morgan and Hunter. It was difficult to be so far away from you three. iii ACKNOWLEDGEMENTS I would like to thank the members of the Li Laboratory for support, discussions, and advice. I will particularly miss lunchtime with the lab! I appreciate the fruitful collaborations with the Bingham Laboratory. Dr. Ivan Sakharov graciously provided the purified windmill palm tree peroxidase, but more than that, he has been a wonderful mentor by discussing peroxidases, and giving invaluable comments on my manuscripts. Dr. David Tabb and his laboratory were very gracious in showing me the ropes in bioinformatics. Thank you to Mr. Sam Fu for consultation about the method for solid-phase reduction of disulfide bonds and for the delicious lunches at McDonald’s. I want to acknowledge Dr. Li and my committee members for their mentorship and guidance throughout this process. Undoubtedly, I could not have done this work without the support and encouragement of my family- my mom, dad, and step-dad; brother, brother-in-law, and sisters; and nephews in Texas. Many thanks to my parents-in-law in Hawaii, who truly make me feel like I’m their daughter. Thanks to my husband, Adam, for always encouraging me and reading my manuscripts. He is a great writer and a brilliant woodworker. Also, Bei Zhang, for running with me- a much needed release of stress! Also, for our discussions on transient expression in tobacco plants- hopefully we will have time to collaborate in the future. I was awarded a fellowship from the USDA National Institute of Food and Agriculture. This project was supported in part by the Agriculture and Food Research Initiative Competitive Grant No. 2012-67011-19671 from the USDA National Institute of Food and Agriculture. iv ABSTRACT Plant secretory peroxidases are important commercial enzymes and play a central role in plant stress responses. The focus of this work is a unique peroxidase from the leaves of a cold tolerant palm, Trachycarpus fortunei, (i.e., windmill palm tree). Like other palm tree peroxidases, windmill palm tree peroxidase is stable at high temperatures and in the presence of denaturants. It is distinct from other palm tree peroxidases in its substrate specificity. The amino acid sequence and glycosylation had not been determined. Because glycosylation is known to play a critical role in plant peroxidase stability and activity, this knowledge is essential for structure activity studies, selection of an expression system for enzyme production, and engineering the enzyme. Glycosylation is a complex modification that is difficult to study; furthermore, there is a lack of analytical tools for characterizing plant glycoproteins. The complete amino acid sequence was determined using cDNA sequencing and biological mass spectrometry. The mature amino acid sequence is 306 residues in length. The presence of a C- terminal signal peptide predicts vacuolar targeting of the enzyme. Native windmill palm tree peroxidase was analyzed at the glycopeptide level to give a qualitative and quantitative assessment of glycosylation at each site. Windmill palm tree peroxidase has 13 sites for N-linked glycosylation, 2 of which are unique. Each site is at least partially occupied by a glycan. Major glycans are paucimannosidic, which supports the assignment of windmill palm tree peroxidase as a vacuolar peroxidase. To carry-out this work, a workflow for analyzing the glycopeptide mass spectrometry data was developed. Included in the workflow are novel tools for glycan database construction, pGlycoFilter, and validation of glycopeptide assignment, gPSMvalidator. New analytical methods are needed for the emerging field of plant glycoproteomics. The novel v methods developed in this dissertation will be useful for the study of other important plant glycoproteins. This knowledge can be used to study the roles of glycosylation in this exceptionally stable and unique palm peroxidase. vi TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iv ABSTRACT .................................................................................................................................... v LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x LIST OF ABBREVIATIONS ........................................................................................................ xi CHAPTER 1. INTRODUCTION ................................................................................................... 1 BACKGROUND ......................................................................................................................... 1 PURPOSE ................................................................................................................................... 2 OBJECTIVES ............................................................................................................................. 3 RATIONALE .............................................................................................................................. 3 CHAPTER 2. AMINO ACID SEQUENCE OF ANIONIC PEROXIDASE FROM THE WINDMILL PALM TREE TRACHYCARPUS FORTUNEI .......................................................... 7 INTRODUCTION ....................................................................................................................... 7 MATERIALS AND METHODS ................................................................................................ 8 RESULTS AND DISCUSSION ............................................................................................... 12 CHAPTER 3. SITE-SPECIFIC N-GLYCOSYLATION AND MICROHETEROGENEITY OF A PEROXIDASE FROM THE WINDMILL PALM TREE TRACHYCARPUS FORTUNEI ......... 24 INTRODUCTION ..................................................................................................................... 24 MATERIALS AND METHODS .............................................................................................. 25 SUPPLEMENTARY DATA .................................................................................................... 28 RESULTS.................................................................................................................................. 28 DISCUSSION ........................................................................................................................... 33 CHAPTER 4. CONCLUSIONS AND FUTURE PERSPECTIVES ............................................ 57 PURPOSE ................................................................................................................................. 57 OBJECTIVES ........................................................................................................................... 57 FUTURE WORK ...................................................................................................................... 60 FUTURE PERSPECTIVES ...................................................................................................... 61 vii APPENDIX A: WORKFLOW FOR ANALYSIS OF PLANT GLYCOPEPTIDE MASS SPECTROMETRY DATA ........................................................................................................... 62 APPENDIX B: CONFIGURATION FILES FOR PROTEOMICS-BASED PROTEIN AND GLYCOPEPTIDE IDENTIFICATION ........................................................................................ 67 APPENDIX C: PLANT N-GLYCAN BIOSYNTHESIS PATHWAY ........................................ 72 APPENDIX D. DESCRIPTION OF SUPPLEMENTARY DATA ............................................. 74 APPENDIX E. GUANIDINATION OF TRYPTIC PEPTIDES WITHOUT DESALTING FOR MATRIX-ASSISTED LASER DESORPTION/IONIZATION-TIME-OF-FLIGHT MASS SPECTROMETRY ANALYSIS .................................................................................................. 76 APPENDIX F. SYNTHESIS OF AN IBERIOTOXIN DERIVATIVE BY CHEMICAL LIGATION: A METHOD FOR IMPROVED YIELDS OF CYSTEINE-RICH SCORPION TOXIN PEPTIDES. ...................................................................................................................... 85 APPENDIX G. CONE SNAIL MILKED VENOM DYNAMICS – A QUANTITATIVE STUDY OF CONUS PURPURASCENS .................................................................................................... 95 APPENDIX H. ANALYSIS OF A CONE SNAIL’S KILLER COCKTAIL – THE MILKED VENOM OF CONUS GEOGRAPHUS ...................................................................................... 108 APPENDIX I. A “CONOVENOMIC” ANALYSIS OF THE MILKED VENOM FROM THE MOLLUSK-HUNTING CONE SNAIL CONUS TEXTILE - THE PHARMACOLOGICAL IMPORTANCE OF POST-TRANSLATIONAL
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