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Chemical Transformations Encoded by a Gene Cluster in Streptomyces Coelicolor Containing an Unusual Gtp Cyclohydrolase

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Chemical Transformations Encoded by a Gene Cluster in Streptomyces Coelicolor Containing an Unusual Gtp Cyclohydrolase Chemical Transformations Encoded by a Streptomyces coelicolor Gene Cluster with an Unusual GTP Cyclohydrolase Item Type text; Electronic Dissertation Authors Spoonamore, James Edward Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 11/10/2021 06:44:52 Link to Item http://hdl.handle.net/10150/194825 CHEMICAL TRANSFORMATIONS ENCODED BY A GENE CLUSTER IN STREPTOMYCES COELICOLOR CONTAINING AN UNUSUAL GTP CYCLOHYDROLASE by James Edward Spoonamore ______________________________ A Dissertation Submitted to the Faculty of the DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOPHYSICS In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2008 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by James Edward Spoonamore entitled Chemical Transformations Encoded by a Gene Cluster in Streptomyces coelicolor Containing an Unusual GTP Cyclohydrolase and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _______________________________________________________________________ Date: April 16, 2008 Vahe Bandarian _______________________________________________________________________ Date:April 16, 2008 Indraneel Ghosh _______________________________________________________________________ Date: April 16, 2008 Nancy Horton _______________________________________________________________________ Date:April 16, 2008 John Osterhout Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: April 16, 2008 Dissertation Director: Vahe Bandarian 3 STATEMENT BY THE AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: James E Spoonamore 4 TABLE OF CONTENTS LIST OF FIGURES ....................................................................................................... 6 ABSTRACT .................................................................................................................... 7 CHAPTER I: INTRODUCTION ................................................................................. 8 Explanation of the problem and its context ............................................................. 8 A review of literature ................................................................................................ 11 Riboflavin biosynthesis and GTP Cyclohydrolase II .............................................. 11 The discovery of vitamins and riboflavin ............................................................... 11 Elucidating the biosynthetic pathway of riboflavin ................................................ 13 The riboflavin gene cluster ..................................................................................... 29 G TP cyclohydrolases in Streptomyces coelicolor A3(2) ......................................... 30 Detailed background about GCH II ........................................................................ 40 An explanation of the Dissertation Format ............................................................ 45 CHAPTER II: PRESENT STUDY............................................................................... 47 SCO 6655 is a non-canonical GTP cyclohydrolase II that catalyzes the transformation of GTP to 2-amino-5-formylamino-6-ribosylamino-4(3H)- pyrimidinone 5©-phosphate (Appendix A) .............................................................. 47 A single amino acid change in the active site of SCO 1441, SCO 2687, and SCO 6655 is sufficient to interchange their activities between canonical and non-canonical (Appendix B) ............................................................................ 48 SCO 6654, a creatininase homolog, catalyzes the transformation of FAPy to APy (Appendix C) ................................................................................................. 49 SCO 6650, a homolog of 6-pyruvoyltetrahydrobiopterin synthase, is a T-fold protein that appears to bind a pyrimidine ring containing substrate (Appendix D) ............................................................................................................. 49 REFERENCES .............................................................................................................. 51 5 TABLE OF CONTENTS - Continued APPENDIX A: SCO 6655 IS A NON-CANONICAL GTP CYCLOHYDROLASE II THAT CATALYZES THE TRANSFORMATION OF GTP TO 2-AMINO-5-FORMYLAMINO-6-RIBOSYLAMINO-4(3H)- PYRIMIDINONE 5©-PHOSPHATE ............................................................................ 63 APPENDIX B: A SINGLE AMINO ACID CHANGE IN THE ACTIVE SITE OF SCO 1441, SCO 2687, AND SCO 6655 IS SUFFICIENT TO INTERCHANGE THEIR ACTIVITIES BETWEEN CANONICAL AND NON-CANONICAL ...................................................................................................... 90 APPENDIX C: SCO 6654, A CREATININASE HOMOLOG, CATALYZES THE TRANSFORMATION OF FAPY TO APY ...................................................... 114 APPENDIX D: SCO 6650, A HOMOLOG OF 6- PYRUVOYLTETRAHYDROPTERIN SYNTHASE, IS A T-FOLD PROTEIN THAT APPEARS TO BIND A PYRIMIDINE RING CONTAINING SUBSTRATE ................................................................................................................ 148 6 LIST OF FIGURES FIGURE 1.1, Riboflavin biosynthetic pathway ............................................................... 16 FIGURE 1.2, Chemical structure of GTP and riboflavin ................................................ 18 FIGURE 1.3, Diacetyl derivation assay .......................................................................... 27 FIGURE 1.4, Predicted GCH II containing transcripts of S. coelicolor A3(2) .............. 34 FIGURE 1.5, Proposed products of the GCH II containing gene clusters ...................... 39 FIGURE 1.6, Mechanism of GCH II from the literature ................................................ 44 7 ABSTRACT Bacterial secondary metabolite biosynthetic pathways are frequently encoded in gene clusters. Genomic sequence information allows the identification of likely biosynthetic clusters based on sequence homology to known proteins. Biochemical characterization of suspected biosynthetic enzymes affords the discovery of pathways which may never be identified by traditional screening approaches. In the work presented here, I, in some cases in collaboration with others, characterize the three intragenomic GTP cyclohydrolase II (GCH II) homologs from Streptomyces coelicolor A3(2) and show that one catalyzes a related but distinct reaction from the other two. The basis for the altered activity is investigated and speaks to the chemical mechanism of not only the unusual enzyme but also to all GCH II enzymes. Further, I investigate two other enzymes found in the same gene cluster as the unusual GCH II. Using biochemical techniques, I show that the product of the unusual GCH II is used as a substrate by a creatinine amidohydrolase homolog. Using structural biology, I show that the third enzyme, a 6- pyruvoyltetrahydropterin synthase (PTPS), can not catalyze the PTPS reaction but is capable of binding a pterin substrate. Finally, I propose that the cluster from S. coelicolor containing the unusual GCH II encodes enzymes for a novel pathway to produce a pterin. 8 CHAPTER I: INTRODUCTION Explanation of the problem and its context Traditionally, bacterial secondary metabolites, such as antibiotics, have been discovered through serendipity by screening fractioned culture media to isolate and identify an active species. Once identified, the biosynthetic route can often be at least partially discerned through judicious use of labeled precursors. Generation of industrially useful over-producing strains are dependent upon successive rounds of random genetic mutation followed by screening. Identifying the metabolite, characterizing its synthesis, and improving the yield of the wild strain each requires an enormous effort and a pinch of luck. For several classes of secondary metabolites, such as polyketides or non-ribosomal peptides, the enzymatic toolkit of bacteria is fairly modular and has been studied sufficiently such that one can reasonably predict the number and kind of proteins, and thus genes, that are needed
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