MIAMI UNIVERSITY The Graduate School Certificate for Approving the Dissertation We hereby approve the Dissertation of Meghan M Holdorf Candidate for the Degree Doctor of Philosophy Advisor______________________________________ Dr. Chris A. Makaroff Reader_______________________________________ Dr. Michael W. Crowder Reader_______________________________________ Dr. Ann E. Hagerman Reader_______________________________________ Dr. Gary A. Lorigan Reader_______________________________________ Dr. John Z. Kiss ABSTRACT CHARACTERIZATION OF ARABIDOPSIS ETHE1, A GENE ASSOCIATED WITH ETHYLMALONIC ENCEPHALOPATHY by M.M.Holdorf Mutations in the ETHE1 gene result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized by symmetric brain lesions, lactic academia, elevated excretion of ethylmalonic acid, and death in the first decade of life. ETHE1-like genes are found in a wide range of organisms; however, to date, a detailed characterization of ETHE1 has not been performed. Therefore, neither the structure nor the function has been established for the enzyme in any organism. In this dissertation, a full structural characterization of the Arabidopsis homolog of ETHE1 as well as information on its functional role in plants is presented. We have obtained the first crystal structure of an ETHE1-like protein as well as performed metal and preliminary substrate analyses providing new insights into the possible role and substrate of ETHE1. In addition, we demonstrate that ETHE1 is essential for both plant growth and development. Characterization of Arabidopsis ETHE1, a Gene Associated With Ethylmalonic Encephalopathy A Dissertation Submitted to the faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry By Meghan Marie Holdorf Miami University Oxford, Ohio 2008 Dissertation Director: Dr. Christopher A. Makaroff Table of Contents Chapter 1 1 Introduction 1 1.1 Ethylmalonic Encephalopathy (EE) 1 1.1.1 Symptoms of Ethylmalonic Encephalopathy 1 1.1.2 Biochemical Markers of EE 1 1.1.3 Ethylmalonic Acid 2 1.1.4 Role of EMA in metabolic pathways 2 1.1.5 Current treatments for EE 4 1.2 Ethylmalonic Encephalopathy Protein 1 (ETHE1) 4 1.3 Metallo-β-lactamase Fold Family of Proteins 6 1.4 The Glyoxalase System 6 1.4.1 The Glyoxalase System 6 1.4.2 Arabidopsis Glyoxalase II enzymes 8 1.5 Hepatoma Subtracted-cDNA Library Clone One 11 1.6 Arabidopsis thaliana 11 1.6.1 Arabidopsis thaliana as a Model system 11 1.6.2 Loss of Function Mutants of Arabidopsis thaliana 13 1.6.3 Embryo Lethal Mutants of Arabidopsis thaliana 13 1.6.4 Seed Development in Arabidopsis thaliana 13 1.7 Sections of the Dissertation 17 1.8 References 19 Chapter 2: Structure of an ETHE1-like Protein from Arabidopsis thaliana 26 2.1 Summary 28 2.2 Introduction 29 2.3 Material and Methods 30 2.3.1 Cloning, Expression, and Purification 30 2.3.2 Crystallization 31 ii 2.3.3 Data Collection 31 2.3.4 Structure Determination and Refinement 31 2.4 Results and Discussion 32 2.4.1 Overall Fold 32 2.4.2 Dimer 34 2.4.3 Metal Binding Site 34 2.4.4 Active Site 40 2.4.5 Structural Basis for Encephalopathy 41 2.4.6 Sequence Analysis 43 2.5 Conclusions 44 2.6 Acknowledgements 45 2.7 References 46 Chapter 3: Spectroscopic Studies of Arabidopsis ETHE1, a Glyoxalase II-like Protein 50 3.1 Summary 51 3.2 Introduction 52 3.3 Material and Methods 54 3.3.1 Over-expression and Purification 54 3.3.2 Extinction Coefficient Determination 54 3.3.3 Metal Analysis 55 3.3.4 Native Molecular Weight Determination 55 3.3.5 Substrate Analysis 55 3.3.6 EPR Spectroscopy 56 3.3.7 1H NMR Spectroscopy 56 3.4 Results 56 3.4.1 Over-expression, Purification, and Characterization of Arabidopsis ETHE1 56 3.4.2 ETHE1 Functions as a Dimer 57 3.4.3 Calculation of the ETHE1 Extinction Coefficient 57 3.4.4 Metal Analysis 60 3.4.5 ETHE1 Does Not Hydrolyze SLG 63 iii 3.4.6 Spectroscopic Studies on ETHE1 64 3.5 Discussion 68 3.6 References 73 Chapter 4: ETHE1, a Gene Associated with Human Ethylmalonic Encephalopathy, is Essential for Endosperm Development in A. thaliana 78 4.1 Summary 80 4.2 Introduction 81 4.3 Material and Methods 84 4.3.1 Plant Material 84 4.3.2 Phylogenetic Analysis of β-lactamase Proteins 84 4.3.3 Molecular Analysis of ETHE1 85 4.3.4 Microscopy 87 4.4 Results 88 4.4.1 Molecular Analysis of ETHE1 88 4.4.2 ETHE1 is Required for Early Seed Development 92 4.4.3 ETHE1 Expression and Localization 99 4.5 Discussion 103 4.5.1 ETHE1 is Essential for Early Endosperm Development 104 4.5.2 Potential Role(s) of ETHE1 106 4.6 Acknowledgements 109 4.7 References 110 Chapter 5: Functional Studies on Arabidopsis Plants and Cell Cultures Expressing an Inducible ETHE1 RNAi 117 5.1 Summary 118 5.2 Introduction 119 5.3 Material and Methods 120 5.3.1 Plant Material 120 5.3.2 Cell Culture 121 iv 5.3.3 Generation of Inducible ETHE1-RNAi Arabidopsis Plants 121 5.3.4 Generation of Inducible ETHE1-RNAi Arabidopsis Suspension cell cultures 121 5.3.5 Molecular Analysis 122 5.3.6 EMA Toxicity studies in Arabidopsis cell culture 122 5.3.7 Microscopy 123 5.4 Results 123 5.4.1 Generation of Transgenic Plants and Suspension Cell Cultures Expressing the Inducible pX7-ETHE1-RNAi Construct 123 5.4.2 Time Course Expression Analysis of px7-ETHE1- RNAi in Cell Cultures 123 5.4.3 ETHE1 is Critical for Arabidopsis Cell Suspension Growth 127 5.4.4 Inducible Expression of ETHE1-RNAi Results in Filamentous Growth and Arrest of Normal Division in Arabidopsis Cell Culture 129 5.4.5 Incubation of Arabidopsis Cell Culture with EMA Undergoes Autophagy 129 5.4.6 ETHE1 is Essential for Plant Germination 132 5.5 Discussion 136 5.5.1ETHE1 is Critical for Cell Survival in Arabidopsis 140 Cell Culture 5.5.2 ETHE1 is Essential for Seed Germination 141 5.6 References 143 Chapter 6: Over-expression of Arabidopsis ETHE1 Results in Enhanced Growth Properties of Plants 147 6.1 Summary 148 6.2 Introduction 149 6.3 Material and Methods 150 v 6.3.1 Plant Material and Growth Conditions 150 6.3.2 Generation of ETHE1-OE plants and cell cultures 152 6.3.3 Microscopy 156 6.3.4 N-terminal Analysis and Protein Localization 156 6.4 Results and Discussion 157 6.4.1 Localization and N-terminal Determination Studies 157 6.4.2 Generation of ETHE1-OE Arabidopsis plants 165 6.4.3 ETHE1256-OE Plants Show Resistance to High of Valine 165 6.4.4 Over-expression of ETHE1256 Leads to Earlier Bolting and Flowering in Arabidopsis 170 6.4.5 ETHE1256 Expression Enhances Seed Yield in Arabidopsis 172 6.4.6 Arabidopsis ETHE1256-OE Plants have Thicker Primary Inflorescence Stems 175 6.4.7 ETHE1256-OE in Nicotiana tabacum Results in Enhanced Growth Properties 177 6.4.8 Concluding remarks 180 6.5 References 182 Chapter 7: Conclusions 190 7.1 Scientific Problems Addressed in this Dissertation 190 7.2 Structural Analysis of ETHE1 in Arabidopsis 190 7.3 Functional Analysis of ETHE1 in Arabidopsis 191 7.4 Potential Roles of ETHE1 193 7.5 Future Aspects and Directions of ETHE1 Analysis 194 7.5.1 Microarray 195 7.5.2 NMR Metabolomics and NMR Metabolite Tracing 195 7.5.3 Inducible pX7-ETHE1-OE Constructs 196 7.5.4 Characterization of Human ETHE1 197 7.6 References 198 vi List of Tables 2.1 Data-Collection and Refinement 33 3.1 Determination of ETHE1’s Extinction Coefficient 62 6.1 Peptide Matches of ETHE1294 Purified Protein 164 vii List of Figures 1.1 Metabolic Routes of EMA Production 3 1.2 Pairwise Alignment of ETHE1 Homologs 5 1.3 Sequence Alignment of Human ETHE1 and Glyoxalase 7 1.4 The Glyoxalase System 9 1.5 Sequence Alignment of GLX2-3(ETHE1) and ETHE1 10 1.6 Sequence Alignment of the Arabidopsis Glyoxalase II isozymes 12 1.7 Life Cycle of Arabidopsis 14 2.1 The At1g53580 monomer 2.2 Sequence Alignment of AtETHE1, ETHE1, GLX2-5, and Human 35 Glyoxalase II (Human GLX2) 36 2.3 The AtETHE1 Dimer 37 2.4 Overlay of the Metal-binding Residues in the AtETHE1 enzyme (Magenta) and the GLX2-5 enzyme (Cyan). 39 2.5 Overlay of the Substrate Binding Residues from the Human Glyoxalase II (Cyan) with the Equivalent Residues in the AtETHE1 Enzyme (Magenta). 42 3.1 Sequence Alignment of ETHE1 and Select Metallo-β-lactamase Fold Proteins 58 3.2 Gel Filtration Elution Profile of Internal Protein Standards and ETHE1 59 3.3 Protein Quantification of ETHE1 Based off of Known Amounts of GLX2-5 61 3.4 1H NMR Spectrum of 3.5 mM Iron Bound ETHE1 at pH 7.2 65 3.5 EPR Spectrum of 4.1 mM ETHE1 under Different Conditions 67 3.6 Dinuclear Metal Binding Center Model for A. thaliana ETHE1 69 4.1 Molecular Characterization of ETHE1 89 4.2 Inactivation of ETHE1 Disrupts Seed Development 93 4.3 Embryo Development in ETHE1(+/-) Siliques 95 4.4 Endosperm Development is Abnormal in ethe1(-/-) Seeds 98 4.5 ETHE1 Expression Patterns 100 viii 4.6 Immunolocalization of ETHE1 in Wild-type Buds 103 5.1 Generation of Inducible RNAi 125 5.2 Time Course Studies of Induced ETHE1-RNAi in Arabidopsis Cells 128 5.3 Effects of ETHE1-RNAi on Cell Culture Growth and Survival 131 5.4 Morphology of Arabidopsis Cell Cultures Expressing ETHE1-RNAi 133 5.5 EMA Toxicity in Arabidopsis Cell Cultures 134 5.6 Inducible ETHE1-RNAi Studies on Seed Germination 137 5.7 Effects of Inducible ETHE1-RNAi on Plant Development 138 6.1 Molecular Analysis of Arabidopsis ETHE1 154 6.2 Localization and Purification of ETHE1256 and ETHE1294 From Transgenic Arabidopsis Cell Cultures 158 6.3 Peptide Mapping of ETHE1294 using MALDI-TOF MS Spectroscopy 161 6.4 Expression Analysis of Transgenic Plants Over-expressing Either ETHE1256 or ETHE1294 166 6.5 Metabolic Routes of EMA Production 168 6.6 Effect of Exogenous Valine on Seedling Growth 171 6.7 The Effect of Over-expressing Either ETHE1256 or ETHE1294 on Plant Growth in Arabidopsis 173 6.8 The Effect of ETHE1256-OE in Arabidopsis on Senescence, Seed Yield, Dry Mass, and Inflorescence Stem Thickness 176 6.9 Effects of Over-expressing Arabidopsis ETHE1256 in Nicotiana Tobacum 178 .