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The HOTHEAD Protein: Assessing Enzymatic Activity Using Computational and Recombinant Protein Expression Approaches

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The HOTHEAD Protein: Assessing Enzymatic Activity Using Computational and Recombinant Protein Expression Approaches The HOTHEAD Protein: Assessing Enzymatic Activity using Computational and Recombinant Protein Expression Approaches By Eric Le Dreff-Kerwin A thesis Presented to the University of Waterloo In fulfillment of the Thesis requirement for the degree of Master of Science In Biology Waterloo, Ontario, Canada, 2019 © Eric Le Dreff-Kerwin 2019 Author’s Declaration I hereby declare that I am the sole author of this thesis. This is a true copy of the thesis, including any required final revisions, as accepted by my examiners. I understand that my thesis may be made electronically available to the public. ii Abstract In Arabidopsis thaliana, a number of genes regulating cuticle synthesis have been identified by virtue of organ fusion phenotype. One such gene, HOTHEAD (HTH) was among those originally identified by this phenotype but its exact role in cuticle formation has proven challenging to determine. Previous bioinformatic work has identified that the HTH protein is a member of the GMC oxidoreductase family, and shares peptide sequence identity with a mandelonitrile lyase and an alcohol dehydrogenase that are within the same protein family. This thesis work investigated the potential enzymatic function of HTH by comparing a structural model to two structural analogs. The structure model of HTH, as determined in this study, shows that HTH shares certain conserved features of GMC proteins. The aim of this research also included isolating a recombinantly expressed HTH protein from Escherichia coli and initial work in Pichia pastoris. Protein isolation attempts in Escherichia coli failed to yield active HTH protein, potentially due to the lack of post-translational modifications. Continuing the work using the eukaryote, Pichia pastoris, should solve the issue. Finally, crude extracts from Arabidopsis thaliana floral tissue were obtained to test for the presence of HTH enzymatic activity but showed no significant mandelonitrile lyase enzymatic activity, potentially due to the inability to successfully extract the HTH protein from the floral tissue. iii Acknowledgements I would like to thank my supervisors, Dr. Susan Lolle and Dr. Todd Holyoak, for all of their help, continuous support and being great examples of great scientists; ones that I always tried to live up to throughout my degree. Even during the most challenging of times, they were always ready to get me back on my feet. Thank you to my committee members, Dr. Barbara Moffatt and Dr. David Rose, for their valuable comments, and for being continuously willing to help advance this project. To the current and past members of the Lolle and Holyoak labs; Dr. Pearl Chang, Therese Francom, Serena Thamilselvan, Saima Hossain, Saeer Adeel, Jackline Ponomariov and Christina Doherty from the Lolle lab, and Matt McLeod, Iain Wallace, Ben Morrow, Norman Tran. Sarah Barwell, Julia Solonenka and Dr. Bidyadhar Das from the Holyoak lab; as well as Nardo Nava-Rodriguez, Nicole Fraser and the other Rose lab members. Thank you all for your support, even if it was one of the many late nights working in the lab. Thank you to Quinn Abram and Dr. Tania Rodriguez-Ramos of the Dixon lab for constantly putting up with my random visits to their lab in search for answers and tips on how to improve my technique. iv Table of Contents Chapter 1 ......................................................................................................................................... 1 General Introduction ....................................................................................................................... 1 1.1 The Plant Cuticle ................................................................................................................................. 2 1.1.1 Cuticle Function .......................................................................................................................................... 2 1.1.2 Cuticle Composition .................................................................................................................................... 2 1.1.3 Biosynthesis Pathway .................................................................................................................................. 3 1.2 HOTHEAD .......................................................................................................................................... 4 1.2.1 The HOTHEAD Gene .................................................................................................................................. 4 1.2.2 HOTHEAD Mutant Alleles.......................................................................................................................... 4 1.2.3 The HOTHEAD Protein .............................................................................................................................. 7 1.2.4 HOTHEAD Protein Localization ................................................................................................................ 8 1.3 Alcohol Dehydrogenase .................................................................................................................... 10 1.4 Mandelonitrile Lyase ......................................................................................................................... 11 1.4.1 The Role of Hydrogen Cyanide in Plants .................................................................................................. 13 1.4.2 HCN & Plant Stress Response .................................................................................................................. 14 1.4.3 HCN and Seed Dormancy ......................................................................................................................... 15 1.4.4 HCN & Nitrate Assimilation ..................................................................................................................... 16 1.4.5 Moonlighting Proteins ............................................................................................................................... 18 1.5 Objectives of the Thesis .................................................................................................................... 19 Chapter 2 ....................................................................................................................................... 20 Structural Analysis of the HOTHEAD Protein Model ................................................................. 20 2.1 Introduction ....................................................................................................................................... 21 2.2 Methods ............................................................................................................................................. 23 2.2.1 HOTHEAD Protein Structural Model Analysis ........................................................................................ 23 2.3 Results & Discussion......................................................................................................................... 24 2.3.1 Hypothesized Enzymatic Function of HTH .............................................................................................. 24 2.3.2 Structural Analogs to the HTH Protein Model .......................................................................................... 24 2.3.3 Conserved Active Site Residues in FAD-Dependent Hydroxynitrle Lyase .............................................. 27 2.3.4 Conserved Active Site Residues in GMC Proteins ................................................................................... 28 2.3.5 Predicted Ligand Binding Site on the HTH Protein Model ...................................................................... 29 2.3.6 Comparing Hydroxynitrile Lyase N-Linked Glycosylation to the HOTHEAD Model ............................ 32 2.4 Conclusions ....................................................................................................................................... 35 Chapter 3 ....................................................................................................................................... 37 Recombinant HOTHEAD Protein Isolation & Enzymatic Assays ............................................... 37 3.1 Introduction ....................................................................................................................................... 38 3.2 Materials ............................................................................................................................................ 39 v 3.3 Methods ............................................................................................................................................. 40 3.3.1 Synthetic HTH cDNA Sequence Design ................................................................................................... 40 3.3.2 Heat Shock Bacteria Transformation ........................................................................................................ 40 3.3.3 Bacterial Plasmid Minipreparation ............................................................................................................ 41 3.3.4 HTH_SUMO Restriction Endonuclease Digestion ................................................................................... 41 3.3.5 HTH Protein Autoinduction Set up ........................................................................................................... 42 3.3.6 Nickel Chromatography of HTH_SUMO Protein ....................................................................................
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