Allosteric regulation of 3 deoxy-D-arabino- heptulosonate 7-phosphate synthase A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at the University of Canterbury by Yifei Fan 2017 Abstract Allostery refers to the process in which interaction of an effector ligand with one site of the protein changes the function of the protein at a distant site. Despite the critical role of allostery in regulation of metabolic pathways, little is known about the details of the allosteric networks and the remarkable diversity in allosteric mechanisms. This study utilises several examples of allosteric proteins to illustrate the interwoven relationships between various allosteric mechanisms, ranging from large conformational changes to subtle dynamic communications. An important metabolic enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), was selected for this study due to its unique diversity of allosteric regulations and its important role as the first committed step in aromatic amino acid biosynthesis. Essential in many pathogenic bacteria, however lacking mammalian counterparts, DAH7PS and related pathway enzymes provide opportunities in development of novel antimicrobial drugs. The first part of this study addresses the determinants of allosteric ligand selectivity and potency for DAH7PS enzymes that exhibit large conformational changes, and provides structural and functional insights that contribute to the understanding of the role of conformational change in allostery. The second part of this study addresses interchangeability between two different allosteric mechanisms by demonstrating the ease of gene fusion to link two contemporary protein domains and produce functional chimera. The third part of this study addresses the allosteric regulation in DAH7PS enzymes from a different subfamily, for which no large conformational changes are involved in delivering allosteric communication. The crystal structure of a related chorismate mutase enzyme contributes to the understanding of protein-protein interactions associated with allosteric regulations employed by this type of I DAH7PS. The final part of this study addresses the current limitations in studying allosteric systems and explores the advantages of new techniques, including Förster resonance energy transfer and electron paramagnetic resonance, in offering valuable information on the timescales and molecular structures associated with allostery. II Deputy Vice-Chancellor’s Office Postgraduate Office Co-Authorship Form This form is to accompany the submission of any thesis that contains research reported in co-authored work that has been published, accepted for publication, or submitted for publication. A copy of this form should be included for each co-authored work that is included in the thesis. Completed forms should be included at the front (after the thesis abstract) of each copy of the thesis submitted for examination and library deposit. Please indicate the chapter/section/pages of this thesis that are extracted from co-authored work and provide details of the publication or submission from the extract comes: Chapter 3 Fan Y, Cross PJ, Jameson GB, Parker EJ: Interchangeable regulatory domains: exploring modular allostery en route to chorismate. Submitted to Proceedings of the National Academy of Sciences 2017. Please detail the nature and extent (%) of contribution by the candidate: 80% The candidate designed and performed the research. The manuscript was drafted by the candidate, and edited and contributed to by co-authors. Certification by Co-authors: If there is more than one co-author then a single co-author can sign on behalf of all The undersigned certifys that: ▪ The above statement correctly reflects the nature and extent of the PhD candidate’s contribution to this co-authored work ▪ In cases where the candidate was the lead author of the co-authored work he or she wrote the text Name: Emily Parker Signature: Date: 11 Oct 2017 III Table of contents Abstract .................................................................................................................................................. I Table of contents .................................................................................................................................IV List of figures .......................................................................................................................................IX List of tables...................................................................................................................................... XIV Abbreviations ................................................................................................................................... XVI Acknowledgements .......................................................................................................................... XIX Chapter 1. Introduction and overview ................................................................................................ 1 1.1. Introduction ...................................................................................................................................... 1 1.1.1. Evolving concept of allostery ............................................................................................... 1 1.1.2. Allostery is structured yet dynamic ....................................................................................... 3 1.2. Aromatic amino acids ...................................................................................................................... 5 1.2.1. Biosynthesis of aromatic amino acids ................................................................................... 5 1.2.2. Regulation of the shikimate pathway .................................................................................... 7 1.2.3. Applications of the shikimate pathway ................................................................................. 8 1.3. DAH7PS ........................................................................................................................................ 10 1.3.1. Classifications and structures of DAH7PS.......................................................................... 10 1.3.2. Allosteric regulation of DAH7PS ....................................................................................... 13 1.4. Regulatory modules ....................................................................................................................... 15 1.4.1. ACT domain ........................................................................................................................ 15 1.4.2. Chorismate mutase .............................................................................................................. 17 1.5. Aims of this thesis .......................................................................................................................... 21 Chapter 2. Allostery and ligand binding in ACT domain-containing DAH7PS ........................... 23 Preface .................................................................................................................................................. 23 2.1. Introduction .................................................................................................................................... 24 2.1.1. Ligand binding .................................................................................................................... 24 2.1.2. Sequence analysis ............................................................................................................... 25 2.2. Choice of mutants .......................................................................................................................... 28 2.3. TmaDAH7PS mutants .................................................................................................................... 29 2.3.1. Preparation of TmaDAH7PS S31V and S31I ..................................................................... 29 2.3.2. Kinetic properties ................................................................................................................ 29 2.3.3. Inhibition ............................................................................................................................. 31 IV 2.4. Characterisation of TyeDAH7PS ................................................................................................... 40 2.4.1. Cloning, expression and purification .................................................................................. 40 2.4.2. Kinetic properties ................................................................................................................ 41 2.4.3. Regulation ........................................................................................................................... 43 2.4.4. Structural characteristics ..................................................................................................... 44 2.5. TyeDAH7PS mutant ....................................................................................................................... 55 2.5.1. Preparation of TyeDAH7PS I31S ........................................................................................ 55 2.5.2. Kinetic properties ................................................................................................................ 55 2.5.3. Inhibition ............................................................................................................................