ENGINEERING AMINOTRANSFERASES FOR THE BIOCATALYTIC PRODUCTION OF AROMATIC D-AMINO ACIDS. CURTIS JAMES WILLIAM WALTON Thesis submitted to the University of Ottawa in partial fulfillment of the requirements for the Doctorate in Philosophy Chemistry Department of Chemistry and Biomolecular Sciences Ottawa-Carleton Chemistry Institute Faculty of Science University of Ottawa © Curtis James William Walton, Ottawa, Canada, 2018 Table of Contents Abstract .......................................................................................................................................... ii Acknowledgements ...................................................................................................................... iii Dedication ..................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Figures .................................................................................................................................x List of Tables .............................................................................................................................. xiii List of Abbreviations ................................................................................................................. xiv Epigraph .................................................................................................................................... xvii Chapter 1. Introduction to D-Amino Acids & Aminotransferases ............................................1 1.1. Amino acids ..........................................................................................................................2 1.1.1. Stereochemistry of amino acids .....................................................................3 1.1.2. Proteinogenic amino acids .............................................................................5 1.2. D-amino acids in the environment ........................................................................................7 1.2.1. Microorganisms .............................................................................................7 1.2.2. Plants and animals..........................................................................................8 1.2.3. Foodstuff and metabolism of D-amino acids .................................................9 1.3. Industrial applications of D-amino acids ............................................................................11 1.3.1. Pharmaceuticals ...........................................................................................11 1.4. Biocatalytic strategies for producing D-amino acids ..........................................................14 1.4.1. Deracemization methods ..............................................................................14 1.4.1.1. Dynamic kinetic resolution .......................................................................15 1.4.1.2. Stereo-inversion ........................................................................................19 1.4.2. Asymmetric synthesis ..................................................................................20 1.5. Pyridoxal 5′-Phosphate Enzymes .......................................................................................22 1.5.1. The identification of the vitamin B6 group..................................................23 1.5.2. The multifarious reactions of PLP ...............................................................24 1.5.3. The Dunathan Stereoelectronic Hypothesis .................................................27 1.5.4. The structural diversity of PLP-dependent enzymes ...................................28 1.6. Aminotransferases ..............................................................................................................31 1.6.1. Classification of ATs ...................................................................................31 1.6.2. Fold-type I ATs ............................................................................................33 v 1.6.2.1. Class I ATs ................................................................................................33 1.6.2.2. Other AT classes .......................................................................................36 1.6.3. Fold-type IV ATs .........................................................................................39 1.6.4. The general mechanism of transamination ..................................................43 1.6.5. Active site topology of ATs .........................................................................45 1.7. Objectives of this thesis ......................................................................................................47 1.8 References ...........................................................................................................................49 Chapter 2. A high-throughput assay for screening L- or D-amino acid specific aminotransferase mutant libraries .............................................................................................55 2.1. Preface ................................................................................................................................56 2.2. Contribution Statement.......................................................................................................56 2.3. Introduction ........................................................................................................................57 2.4 Results and Discussion ........................................................................................................59 2.4.1. Screening assay development ......................................................................59 2.4.2. Screening of site saturation mutagenesis libraries .......................................69 2.4.3. Substrate specificity screening .....................................................................73 2.4.4. Implementation of our assay in directed evolution ......................................78 2.5. Conclusion ..........................................................................................................................79 2.6. Acknowledgements ............................................................................................................79 2.7. Materials and Methods .......................................................................................................80 2.7.1. Materials ......................................................................................................80 2.7.2. Mutagenesis .................................................................................................80 2.7.3. Preparation of clarified cell lysate ...............................................................81 2.7.4. Protein expression and purification .............................................................81 2.7.5. Screening assays ..........................................................................................82 2.7.6. Steady-state kinetics.....................................................................................83 2.8. References ..........................................................................................................................84 Chapter 3. Engineered aminotransferase for the production of D-phenylalanine derivatives using biocatalytic cascades ..........................................................................................................86 3.1. Preface ................................................................................................................................87 3.2. Contribution Statement.......................................................................................................87 vi 3.3. Introduction ........................................................................................................................88 3.4. Results and Discussion .......................................................................................................91 3.5. Conclusion ........................................................................................................................102 3.6. Acknowledgements ..........................................................................................................103 3.7. Supplemental Information ................................................................................................103 3.7.1. Supplementary results ................................................................................103 3.7.1.1. DDO assay development.........................................................................103 3.7.1.2. Supplementary tables ..............................................................................106 3.7.1.3. Supplementary scheme and figures ........................................................108 3.7.2. Materials ....................................................................................................113 3.7.3. Chemicals ...................................................................................................113 3.7.4.