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Proquest Dissertations INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not serxt UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to Ise removed, a note vrill indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are availat>le for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 UMI BIOCHEMICAL ANALYSIS OF GLVCOSYLTRANSFERASES INVOLVED IN DOXORUBICIN BIOSYNTHESIS BY Streptomyces sp. strain C5 DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Anton James Woo, B. S. The Ohio State University 2000 Dissertation Committee: Approved by Dr. William Strohl Dr. Tina Henkin, Adviser Dr. Charles Daniels Adviser Department of Microbiology Dr. John Reeve UMI Number 9983010 UMI UMI Microform9983010 Copyright 2000 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 ABSTRACT Daunorubicin (daunomycin) and doxorubicin (Adriamycin) are clinically important anthracycline antitumor agents produced by Streptomyces sp. strain C5 and Streptomyces peucetius ATCC 29050. Most anthracyclinones, or anthracyclines lacking a sugar moiety, are biologically inactive, which underlies the critical nature for glycosylation in the biosynthesis of these compounds (Arcamone, 1981 b). Unfortunately, extensive characterizations of glycosyltransferases (GTs) involved in antibiotic biosynthesis have not been prevalent, primarily due to the limited availability of the often complex carbohydrates required. Recent findings on structure and mechanistic implications have arisen strictly from sequence analysis and hydrophobic cluster analysis (HCA) (Saxena et ai, 1995; Fish and Cundliffe, 1997; Kapitonov and Yu, 1999). Within the Streptomyces sp. stain C5 and Streptomyces peucetius daunorubicin/doxorubicin biosynthetic gene clusters are two putative glycosyltransferase- encoding genes: dnmS and dauH. Biosynthesis of daunorubicin requires one glycosyltransferase-mediated step, believed to be the addition of TDP-daunosmine to e- rhodomycinone forming rhodomycin D. Additionally, higher glycosides of daunorubicin are produced by both Streptomyces species, implicating the need for a second glycosyltransferase activity. I describe here the cloning, overexpression, purification, and analysis of both glycosyltransferases from Streptomyces sp. stain C5. Only DnmS demonstrated the ability to glycosylate e-rhodomycinone to rhodomycin D. The sugar source used in these assays was a strain C5 mutant with only a functional TDP-daunosamine biosynthetic pathway. Kinetic binding data also supported DnmS as the GT responsible for the formation of rhodomycin D. Moreover, analysis of DnmS binding to the different substrates and products allowed for a reaction mechanism to be proposed. Finally, the generation of site-directed mutants of DnmS addressed structure-function relationships, particularly of conserved amino acids involved in substrate binding. Ill DEDICATION To my wife Angie, who represents ail that is wonderful and loving in this life, and to our son, Nathan. IV ACKNOWLEDGMENTS I wish to thank my adviser. Dr. William Strohl, for his supportive and rational guidance throughout my graduate school career and for his contagious enthusiasm toward scientific discovery and education. I am also deeply indebted to Dr. Nigel Priestley, who supported and enhanced my research greatly and helped me with many technical aspects of my experiments. Additionally, many thanks are in order for Dr. Tyrrell Conway and Dr. Tina Henkin for serving as “surrogate” advisors near the end of my dissertation work. I would also like to thank Dr. Brian Ahmer for temporary housing and the Department of Microbiology for financial support during my last year. I wish to thank former members of the Strohl laboratory. Dr. Michael Dickens, Dr. Yun Li, Dr. Vineet Rajgarhia, Chuck DeSanti, and Rob Walczak. Their instruction and input were essential to my professional development and their fnendships are invaluable. Moreover, I would like to thank Dr. Trevor Darcy for years of stimulating discussion, scientific and non-scientific, and Don Ordaz and Jon-David Sears for particularly helping with all of the “little things” . I also wish to thank Dr. Merv Bibb for graciously providing the streptomyces expression vectors and Dr. Ben Liu for help with chemical synthesis concerns. My family, Angie’s family, and all of our friends receive my full appreciation for love, encouragement, and support throughout my graduate studies. In particular, I wish to thank my father. Dr James Woo, who planted the seed so many years ago. Thus begins the harvest... Finally, “to Him who sits on the throne, and to the Lamb, be praise, honor, glory, and power, forever and ever. Amen.” VI VITA July 9, 1971......................................................Bom - Mayfield Heights, Ohio 1993................................................................... B S. Biology, Bowling Green State University, Bowling Green, Ohio 1993-present ......................................................Graduate Teaching and Research Associate, The Ohio State University, Columbus, Ohio PUBLICATIONS Research Publications 1. Walczak, R.J., A J. Woo, W.R. Strohl, and N. D. Priestley. 2000. Nonactin biosynthesis; The potential nonactin biosynthesis gene cluster contains type II polyketide-synthetase-like genes. FEMS Microbiology Letters 183:171 -175. 2. Woo, A. J., W. R. Strohl, and N. D. Priestley. 1999. Nonactin biosynthesis: The product of nonS catalyzes the formation of the furan ring of nonactic acid. Antimicrobial Agents and Chemotherapy 43:1662-1668. 3. Strohl, W. R , Dickens, M. L , Rajgarhia, V., Woo, A., and N. Priestley. Anthracyclines. In. Strohl WR, ed. Biotechnology of Industrial Antibiotics, 2nd ed. New York: Marcel Dekker, Inc, 1997:577-657. 4. Strohl, W. R., Dickens, M. L , Rajgarhia, V., Walczak, R., Woo, A., and N. D. Priestley. 1997. Biochemistry, molecular biology and protein - protein interactions in daunorubicin/doxorubicin biosynthesis: Proceedings of the Biotechnology of Microbial Products (BMP 1997). C. R. Hutchinson and J. McAlpine, eds. Developments in Industrial Microbiology. 35: 15-22. FIELDS OF STUDY Major Field: Microbiology VII TABLE OF CONTENTS Page Abstract .......................................................................................................................... ii Dedication ...................................................................................................................... iv Acknowledgements ....................................................................................................... v V ita.................................................................................................................................. vii Table of Contents ........................................................................................................... viii List of Tables ................................................................................................................... xiii List of Figures ................................................................................................................. xiv Chapters; 1. Introduction ............................................................................................................... I Historical significance ........................................................................................ 1 Daunorubicin and doxorubicin producing Streptomyces spp ........................... 5 Daunorubicin and doxorubicin mechanism of action ...................................... 6 Toxicity associated with doxorubicin therapy .................................................. 7 Biosynthetic pathway for daunorubicin/doxorubicin production .................... 8 Polyketide synthase-catalyzed aklanonic acid biosynthesis ............................. 8 Formation of e-rhodomycinone from aklanonic acid ....................................... 13 Evidence that e-rhodomycinone is the last aglycone intermediate ................. 17 TDP-daunosamine biosynthesis ......................................................................... 18 v iii Page Conversion of rhodomycin D to doxorubicin ............................................... 22 Higher glycosides
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