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Copyright by Anthony James Romo 2016 The Dissertation Committee for Anthony James Romo Certifies that this is the approved version of the following dissertation: Discovery of the Amipurimycin and Miharamycins Biosynthetic Gene Clusters and Insight into the Biosynthesis of Nogalamycin Committee: Hung-wen (Ben) Liu, Supervisor Walter L. Fast Adrian T. Keatinge-Clay Sean M. Kerwin Christian P. Whitman Discovery of the Amipurimycin and Miharamycins Biosynthetic Gene Clusters and Insight into the Biosynthesis of Nogalamycin by Anthony James Romo, Pharm.D. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May 2016 Dedication I would like to dedicate this work to my amazingly talented wife, Dr. Dawn Kim- Romo, and our beautiful son, Julian Romo. Thank you for encouraging me to continue when I wanted to quit and for giving me the inspiration to reach farther than my grasp. "O sol che sani ogne vista turbata, tu mi contenti sì quando tu solvi, che, non men che saver, dubbiar m'aggrata.” (Dante, Inferno, 11.91-93) Acknowledgements First of all, I want to thank God for my wife and son and for the strength to get through my graduate studies, for, though it is often said at this point in a graduate student’s life, there were times when I really could never imagine finishing graduate school! I would be completely and unforgivably remiss if I did not thank the people who have helped shape my experience. I thank Dr. Zhiwen (Jonathan) Zhang for giving me my first experience in medicinal chemistry research and Dr. Gabby Moreno-Thibodeaux for being my first mentor. Next, I would like to thank Dr. Hung-wen (Ben) Liu for allowing me to work in and ultimately study in his lab, and Dr. Yung-nan Liu for greatly facilitating my progress. Not only was Dr. Liu a knowledgeable and thoughtful advisor, but my time in his lab allowed me to interact with a vibrant and enthusiastic group of graduate students and postdocs, some of whom have made a lifelong impression on me. I would like to specifically thank Dr. Yasushi Ogasawara for giving me the set of skills that were instrumental to my work; Aoshu Zhong for being an apt protégé and skillful teacher; Geng-Min Lin for his patient guidance and encouragement, as well as for continuing my work; Dr. Chia-I Lin for her advice and expertise which often helped me to think more deeply about my results; and finally, Dr. Mark Ruszczycky for telling me to stop “thinking like a first year graduate student” and for being one of the most thorough editors I have ever met. I will never forget my time in the Liu Lab, and I am glad to join the brilliant alumni who have studied with Dr. Liu. Finally, I would like to thank my committee members for their patience and commitment to this endeavor. v Discovery of the Amipurimycin and Miharamycins Biosynthetic Gene Clusters and Insight into the Biosynthesis of Nogalamycin Anthony James Romo, Ph.D. The University of Texas at Austin, 2016 Supervisor: Hung-wen (Ben) Liu Advancements in our ability to obtain high quality bacterial whole genome sequences have increased the rate natural product biosynthetic gene clusters are identified, but these projects require computational methods for gene annotation which are limited by their reliance on comparison to previously annotated genes. Thus, even in a well-defined cluster with a known product it may be difficult to predict how structural characteristics arise if they have no known precedent. Without further work, such limitations will continue to impede the utility of sequenced bacterial genomes. Biosyntheses of the peptidyl nucleoside antibiotics and atypical anthracyclines like nogalamycin are topics which exemplify these challenges. The peptidyl nucleoside antibiotics (PNAs) are a structurally complex group of natural products with diverse biological activities which could be useful for the development of novel antimicrobials. Amipurimycin and the miharamycins are remarkably similar PNAs elaborated by the bacteria Streptomyces miharaensis ATCC 19440 and Streptomyces novoguineensis CBS 199.78, respectively. Their dissimilarity to other well-characterized groups of antibiotics has presented a challenge to the study of their unique structural features. Herein, we describe the identification of the amipurimycin and miharamycins biosynthetic gene clusters though a comparative genomics approach. vi Besides providing insight into the biosynthesis of the rare amino acids adorning these PNA, our analysis revealed a plausible biosynthetic route to the unique 2-aminopurine nucleobase and suggests the core saccharides are generated by a polyketide synthase. The anthracyclines are a mainstay of chemotherapy, but their use is limited by fatal cardiotoxicity and tumor resistance. The structures of the anthracyclines are sensitive to modification, as even small changes can ablate their biological activity. Nevertheless, the diversification of anthracyclines through semi- or total syntheses is an ongoing effort. Nogalamycin is rare amongst the anthracyclines because of its extended ring system and unusual glycosylation pattern. It is hoped an understanding of the biosynthesis of nogalamycin could allow the incorporation of its uncommon structural features into other anthracyclines to develop novel compounds with improved actitivty. Towards this end, we investigated the biosynthesis of the amino sugar found in nogalamycin, nogalamine, to clarify ambiguous steps in the reported biosynthetic pathway for this sugar. vii Table of Contents List of Tables ....................................................................................................... xiii List of Figures ...................................................................................................... xiv Chapter 1: Biosynthesis of The Peptidyl Nucleoside Antibiotics and Glycosylation of Anthracyclines, Under-Explored Topics in the Study of Natural Products ....1 1.1. INTRODUCTION ........................................................................................1 1.2. BIOSYNTHESIS OF THE PEPTIDYL NUCLEOSIDE ANTIBIOTICS ....3 1.2.1. Introduction ......................................................................................3 1.2.2. Amino Acid Attachment ..................................................................5 1.2.2.1. ATP-dependent Peptide Bond Formation ....................................7 1.2.2.1.1. ATP-grasp Ligases ...............................................................7 Blasticidin S ..................................................................................8 Polyoxins and Nikkomycins .........................................................9 Albomycins .................................................................................11 A201A .........................................................................................13 1.2.2.1.2. Non-Ribosomal Peptide Synthetases ..................................14 Muraymycins ..............................................................................15 Pacidamycins ..............................................................................17 Streptothricins .............................................................................20 1.2.2.2. ATP-independent Peptide Bond Formation ...............................23 1.2.2.2.1. N-acylation .........................................................................23 Amicetins ....................................................................................23 Gougerotin ..................................................................................25 1.2.2.2.2. Miscellaneous .....................................................................27 Pacidamycins ..............................................................................27 A-503083 ....................................................................................28 Muraymycins ..............................................................................29 1.2.2.3. Unknown Mechanism ................................................................30 Blasticidin S and Mildiomycin ...................................................30 viii Puromycin ...................................................................................31 1.2.3. Core Saccharide Origin: Beyond Hexoses Versus Pentoses ..........32 1.2.3.1. Incidental....................................................................................32 Puromycin and A201A ...............................................................33 Pacidamycins ..............................................................................34 1.2.3.2. Intact ..........................................................................................36 Blasticidin S, Arginomycin, and Gougerotin ..............................36 Streptothricins .............................................................................40 Amicetins ....................................................................................42 1.2.3.3. Built-up ......................................................................................43 Polyoxins and Nikkomycins .......................................................43 Mildiomycin ................................................................................44 Albomycins .................................................................................45 A-503083s ...................................................................................47