Molecular and structural characterization of the candidate enzymes responsible for tartaric acid synthesis in the grapevine By Yong Jia Thesis by a combination of conventional and publication formats submitted to The University of Adelaide for the degree of Doctor of Philosophy The University of Adelaide School of Agriculture Food and Wine November 2015 1 Table of Contents Abstract ............................................................................................................................................ 5 Declaration ....................................................................................................................................... 8 Statement of the contributions of jointly authored papers and manuscripts ............................ 9 Acknowledgement ......................................................................................................................... 10 Chapter 1 Introduction ................................................................................................................. 11 1.1 General introduction ........................................................................................................... 12 1.2 The chemistry of tartaric acid ............................................................................................ 16 1.3 Unequal distribution in higher plants ............................................................................... 18 1.4 Tartaric acid in grape and wine ......................................................................................... 21 1.5 Industrial application and production .............................................................................. 21 1.6 Tartaric acid biosynthesis ................................................................................................... 23 1.6.1 The primary TA biosynthetic pathway in grapevine ................................................. 23 1.6.2 TA biosynthetic pathway in Geraniaceae plants ........................................................ 26 1.6.3 TA biosynthetic pathway identified in Legumaceae plants ....................................... 27 1.7 Enzymes responsible for tartaric acid biosynthesis ......................................................... 28 1.8 Gene duplication, protein structural variation and plant phenotypic diversity ............ 31 1.9 Protein structural variation and biological function ........................................................ 33 1.10 Conclusion and aims ......................................................................................................... 34 Chapter 2 New insights into the evolutionary history of plant sorbitol dehydrogenase ......... 38 Chapter 3 Amino acid substitutions at catalytic site of plant sorbitol dehydrogenase lead to a shift in substrate specificity .......................................................................................................... 39 3.1 Introduction ......................................................................................................................... 40 3.2 Methods ................................................................................................................................ 44 3.2.1 Chemicals ...................................................................................................................... 44 3.2.2 Sequence alignment and phylogenetic analyses......................................................... 44 3.2.3 Molecular cloning and transformation ...................................................................... 45 3.2.4 Site-directed mutagenesis ............................................................................................ 46 3.2.5 Recombinant protein purification .............................................................................. 46 3.2.6 Enzyme assay ................................................................................................................ 47 3.3 Results and discussion ........................................................................................................ 48 3.3.1 Bioinformatic analyses ................................................................................................. 48 3.3.2 Molecular cloning and recombinant protein purification ........................................ 51 2 3.3.3 Enzymatic substrate specificity of SDHs .................................................................... 56 3.4 Conclusion........................................................................................................................ 60 3.5 Abbreviations ................................................................................................................... 60 Chapter 4 Identification and structural characterization of a putative 2-keto-L-gulonic acid reductase from Vitis vinifera ......................................................................................................... 62 4.1 Abstract ................................................................................................................................ 64 4.2 Introduction ......................................................................................................................... 64 4.3 Results and Discussion ........................................................................................................ 68 4.3.1 Molecular characteristics of the putative 2-keto-L-gulonate reductase .................. 68 4.3.2 Enzymatic activity of VV2KR ..................................................................................... 71 4.3.3 Crystallization .............................................................................................................. 72 4.3.4 Overall structure .......................................................................................................... 74 4.3.5 Dimer interface ............................................................................................................. 79 4.3.6 Co-enzyme binding ...................................................................................................... 81 4.3.7 The catalytic site of VV2KR and substrate binding .................................................. 86 4.3.8 Substrate docking and substrate specificity ............................................................... 92 4.3.9 The potential role of VV2KR in TA biosynthesis in the grapevine .......................... 96 4.4 Conclusion ........................................................................................................................... 98 4.5 Materials and Methods ....................................................................................................... 98 4.5.1 Identification of a putative 2-keto-L-gulonate reductase gene from grapevine ...... 98 4.5.2 Molecular cloning ......................................................................................................... 99 4.5.3 Recombinant protein expression and purification .................................................... 99 4.5.4 Enzyme kinetic assays ................................................................................................ 100 4.5.5 Protein crystallization ................................................................................................ 101 4.5.6 Data collection, structure determination and refinement ....................................... 101 4.5.7 Substrate docking ....................................................................................................... 102 4.6 Supplementary Figures ..................................................................................................... 103 4.7 Contributions ..................................................................................................................... 107 4.8 Acknowledgement ............................................................................................................. 107 4.9 Abbreviations ..................................................................................................................... 107 Chapter 5 Summary, Future Directions and Conclusions ....................................................... 113 5.1 Summary of Research Outcomes ..................................................................................... 113 5.1.1 Evolutionary origin of the grapevine LIDH ............................................................ 113 5.1.2 Substrate specificity of SDH and its effect on TA biosynthesis in higher plants ... 114 3 5.1.3 Enzymatic and protein structural characterization of VV2KR in grapevine ....... 115 5.2 Discussion and Future Research ...................................................................................... 117 5.2.1 The biological function of the Class II SDHs and TA biosynthesis in higher plants .............................................................................................................................................. 117 5.2.2 In vivo characterization of the putative function of VV2KR in TA biosynthesis in the grapevine ....................................................................................................................... 120 5.2.3 Identification of the other candidate enzymes involved TA biosynthesis pathway .............................................................................................................................................. 123 5.3 Conclusion ........................................................................................................................