UC San Diego UC San Diego Electronic Theses and Dissertations Title Manipulating posttranslational modification in natural product biosynthesis Permalink https://escholarship.org/uc/item/0sn8v44w Author Foley, Timothy Leyden Publication Date 2010 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Manipulating Posttranslational Modification in Natural Product Biosynthesis A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Chemistry by Timothy Leyden Foley Committee in charge: Professor Michael D. Burkart, Chair Professor Stuart Brody Professor Elizabeth A. Komives Professor Emmanuel Theodorakis Professor Jerry Yang 2010 Copyright Timothy Leyden Foley, 2010 All rights reserved. The dissertation of Timothy Leyden Foley is approved, and it is acceptable in quality and form for publication on microfilm and electronically _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Chair University of California, San Diego 2010 iii DEDICATION This dissertation is dedicated to my family and friends. Most of all, to my mother, whose constant support and unwaivering acceptance of my decisions has been the cornerstone to my success, now and always. To my father, who bestowed upon me the desire for intellectual consummation and a curiousity about the physical world from an early age. To my brother, whose comradery and competition for attention at a young age allowed me to develope a drive for acheivement. To my godparents, Ramona and Herb Gorecki, who have always been there as my surrogate extended family. This dissertation is also dedicated to Erin Mesplou. Your love and support have provided me with the courage to complete my studies, without which this work would not have been possible. iv EPIGRAPH Very often, in the course of scientific experimentation, a totally unexpected observation is made that is either unrelated or only incidentally related to the problem under immediate investigation. When it captures the attention of an alert, inquisitive mind, this observation may open the door for an entirely new study that is often more fruitful than that of the original design. Earl Stadtman v TABLE OF CONTENTS DEDICATION ..............................................................................................................iv EPIGRAPH.....................................................................................................................v TABLE OF CONTENTS ..............................................................................................vi LIST OF ABBREVIATIONS ........................................................................................x LIST OF FIGURES......................................................................................................xii LIST OF TABLES .......................................................................................................xv ACKNOWLEDGEMENTS ........................................................................................xvi VITA..........................................................................................................................xviii ABSTRACT OF THE DISSERTATION...................................................................xxii Chapter 1 INTRODUCTION........................................................................................1 NATURAL PRODUCTS........................................................................................2 NONRIBOSOMAL PEPTIDES AND THEIR BIOSYNTHESIS .........................8 THIOTEMPLATE LOGIC...................................................................................11 THIOESTER INTERMEDIATES........................................................................12 POSTTRANSLATIONAL MODIFICATION OF CP DOMAINS .....................13 PHOSPHOPANTETHEINYL TRANSFERASE ENZYMES .............................15 BIOMEDICAL RELEVANCE.............................................................................17 PK AND NRP COMPOUNDS AS VIRULENCE FACTORS ............................18 DEVELOPMENT OF A CP-DOMAIN ISOLATION TECHNIQUE .................19 TARGET ISOLATION PROCEDURE AND COMPLICATION.......................24 vi Chapter 2 SITE-SPECIFIC PROTEIN MODIFICATION: ADVANCES AND APPLICATIONS..........................................................................................................27 ABSTRACT..........................................................................................................28 INTRODUCTION ................................................................................................28 BIOCHEMICAL (in vitro) TOOLS......................................................................30 PROTEOMIC (ex vivo) TOOLS ..........................................................................34 CELLULAR IMAGING/RECOMBINANT TOOLS...........................................38 CONCLUSION.....................................................................................................42 ACKNOWLEDGEMENTS..................................................................................45 Chapter 3 A HOMOGENOUS RESONANCE ENERGY TRANSFER ASSAY FOR PHOSPHOPANTETHIENYL TRANSFERASE.........................................................46 ABSTRACT..........................................................................................................47 INTRODUCTION ................................................................................................47 MATERIALS & METHODS ...............................................................................50 RESULTS & DISCUSSION.................................................................................54 CONCLUSIONS...................................................................................................73 ACKNOWLEDGEMENTS..................................................................................73 Chapter 4 PHOSPHOPANTETHEINYL TRANSFERASE INHIBITORS AND SECONDARY METABOLISM ..................................................................................74 ABSTRACT..........................................................................................................75 INTRODUCTION ................................................................................................75 RESULTS & DISCUSSION.................................................................................79 vii CONCLUSIONS...................................................................................................93 MATERIALS AND METHODS..........................................................................94 ACKNOWLEDGEMENTS................................................................................110 Chapter 5 A STRATEGY TO DISCOVER INHIBITORS OF BACILLUS SUBTILIS SURFACTIN-TYPE PHOSPHOPANTETHEINYL TRANSFERASE..112 ABSTRACT........................................................................................................113 INTRODUCTION ..............................................................................................114 MATERIALS AND METHODS........................................................................118 RESULTS ...........................................................................................................126 DISCUSSION .....................................................................................................139 ACKNOWLEDGEMENTS................................................................................144 Chapter 6 PREPARATION OF FRET REPORTERS TO SUPPORT CHEMICAL PROBE DEVELOPMENT.........................................................................................146 ABSTRACT........................................................................................................147 INTRODUCTION ..............................................................................................147 RESULTS AND DISCUSSION .........................................................................150 MATERIALS AND METHODS........................................................................156 ACKNOWLEDGEMENTS................................................................................165 Chapter 7 INHIBITORS OF SURFACTIN-TYPE PHOSPHOPANTETHEINYL TRANSFERASE IDENTIFIED BY QUANTITATIVE HIGH THROUGHPUT SCREENING..............................................................................................................167 ABSTRACT........................................................................................................168 viii INTRODUCTION ..............................................................................................168 RESULTS ...........................................................................................................171 CONCLUSIONS.................................................................................................233 ACKNOWLEDGEMENTS................................................................................233 REFERENCES .........................................................................................................234 ix LIST OF ABBREVIATIONS 4’-PP 4’-phosphopantetheine ACP acyl carrier protein AT acyl transferase