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Biosynthetic Mechanism of the Antibiotic Capuramycin

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University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2018 BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN Erfu Yan University of Kentucky, [email protected] Author ORCID Identifier: https://orcid.org/0000-0002-5545-0995 Digital Object Identifier: https://doi.org/10.13023/etd.2018.360 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Yan, Erfu, "BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN" (2018). Theses and Dissertations--Pharmacy. 92. https://uknowledge.uky.edu/pharmacy_etds/92 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Erfu Yan, Student Dr. Steven Van Lanen, Major Professor Dr. David Feola, Director of Graduate Studies BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Erfu Yan Lexington, Kentucky Director: Dr. Steven Van Lanen, Professor of Pharmaceutical Science Lexington, Kentucky 2018 Copyright © Erfu Yan 2018 ABSTRACT OF DISSERTATION BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN A-102395 is a member of the capuramycin family of antibiotics which was isolated from the culture broth of Amycolatopsis sp. SANK 60206. A-102339 is structurally classified as a nucleoside antibiotic, which like all members of the capuramycin family, inhibits bacterial MraY (translocase I) with IC50 of 11 nM which is the lowest among the capuramycin family. A semisynthetic derivative of capuramycin is currently in clinical trials as an antituberculosis antibiotic, suggesting high potential for using A- 102395 as a starting point for new antibiotic discovery. In contrast to other capuramycins, A-102395 has a unique arylamine-containing polyamide side chain. The biosynthetic gene cluster of A-102395 was previously identified and includes 35 putative open reading frames responsible for biosynthesis and resistance. Presently, there are no reports focused on the biosynthesis of this polyamide chain. Here we present the functional assignment and biochemical characterization of seven proteins, Cpr33-38 and Cpr12, that initiate the biosynthesis of the polyamide. Functional characterization of Cpr38, which has sequence similarity to the gene products encoded by pabA and pabB from E. coli, revealed that it functions as a 4- amino-4-deoxychorismate (ADC) synthase catalyzing a two-step reaction involving amidohydrolysis of L-Gln with ammonia channeled and incorporated into chorismic acid to generate ADC. Cpr12, encoded by a gene that was originally proposed to be outside the gene cluster and sharing similarity to proteins annotated as ADC lyase, was revealed to catalyze the elimination of pyruvate to form PABA. Cpr36 is demonstrated to function as a free-standingpeptidyl carrier protein (PCP), which is activated to form holo-protein from the apo-form. Cpr37, which belongs to the adenylation domain protein in the nonribosomal peptide synthase (NRPS), subsequently activates PABA and loads it to holo-Cpr36 Two proteins Cpr34 and Cpr35 work in concert to catalyze decarboxylative condensation between a thioester linked PABA and malonyl-S-acyl carrier protein (ACP) during aromatic polyketide biosynthesis catalyzed by type II polyketide synthases. Following condensation, Cpr33 acts as 3-oxoacyl-ACP reductase that catalyzes reduction to the β-hydroxythioester intermediate. In this scenario, hydride is predicted to be added to the re face to generate the S configuration resulting in the same stereochemical outcome as other 3-oxoacyl-ACP reductase (FabG) from bacterial type II fatty acid synthases.These findings are critical advancement for interrogating the biosynthesis of the unusual chemical components of the family of antibiotics of capuramycin. KEWWORDS: capuramycins, peptidoglycan cell wall, polyamide assembly Erfu Yan Student’s Signature 08/28/2018 Date BIOSYNTHETIC MECHANISM OF THE ANTIBIOTIC CAPURAMYCIN By Erfu Yan Steven Van Lanen Director of Dissertation David Feola Director of Graduate Studies 08/28/2018 Date 献给我的妻子和女儿，婷婷和雯潇，谢谢你们一路相濡以沫 This dissertation is dedicated to my dear wife and daughter, Tingting and Liliana, whose love and support made this journey through graduate school possible. ACKNOWLEDGMENTS I would like to take this special moment to express my gratitude to my mentor, Dr. Steven Van Lanen, for his guidance, support, and inspiration throughout my study in his group. I am grateful for the chance to seek refuge in this lab following departure of Dr. Guo’s lab in my second PhD year. His expertise and insightful discussions have benefited me greatly and extensively. I would also like to thank my committee members, Dr. Chang-guo Zhan, Dr. Oleg Tsodikov, and Dr. Chris Richards, for their invaluable advice and comments over the years, and I would like to sincerely thank Dr. Zhenyu Li for being the external department examiner for my dissertation defense. Special thanks to all current and previous members from the Van Lanen group: Dr., Dr. Zhaoyong Yang, Dr. Sandra Barnard, Dr. Xiuling Chi, Dr. Anwesha Goswami, Dr. Xiaodong Liu, Dr. Zheng Cui, Dr. Ying Huang, Matthew McErlean, Ashley Arlinghaus, and Jonathon Overbay. Also, a special thanks to Dr. Bert C. Lynn and Dr. Fang Huang in Department of Chemistry, University of Kentucky. I would also like to thank Dr. Minakshi Bhardwaj for analyzing all the NMR data showed in this thesis. It was a great honor to work with and learn from you all! Last but not least, I would like to acknowledge my family and friends for their constant support and encouragement. I need to thank all my friends and coworkers who have been my teachers in different aspects of my life in graduate school. iii TABLE OF CONTENTS ACKNOWLEDGMENTS ........................................................................................... iii LIST OF TABLES ....................................................................................................... vii LIST OF FIGURES ................................................................................................... viii LIST OF ABBREVIATIONS ....................................................................................... xi Chapter 1: Introduction and Background ....................................................................... 1 1.1 Natural Products – Significance ........................................................................... 1 1.2 Need for New Anti-tuberculosis Antibiotics ........................................................ 2 1.3 Biosynthesis of Peptidoglycan Cell Wall ............................................................. 4 1.4 MraY – Structure and Function ........................................................................... 7 1.5 Inhibitors of MraY-Translocase I ....................................................................... 12 1.6 Non-ribosomal Peptides and Their Synthesis .................................................... 15 1.7 Mass Spectrometry and Protein Analysis .......................................................... 18 1.8 Discovery of Capuramycins ............................................................................... 23 1.9 Current Understanding of the Biosynthetic Pathways of A-102395 .................. 26 1.10 Aims of this Study ............................................................................................ 29 Chapter 2: Elucidating the mechanism of Cpr38 and Cpr12 ....................................... 30 2.1 Background ........................................................................................................ 30 2.2 Materials and Methods ......................................................................................
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