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Characterization of Nonr, an Esterase That Confers Nonactin Resistance

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Characterization of Nonr, an Esterase That Confers Nonactin Resistance CHARACTERIZATION OF NONR, AN ESTERASE THAT CONFERS NONACTIN RESISTANCE A Thesis Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By James E. Cox, B.S., B.A. ***** The Ohio State University 2004 Dissertation Committee: Approved by Professor Robert W. Brueggemeier, Adviser Professor Larry W. Robertson Professor Lane Wallace _ _______________________ Adviser Professor Nigel D. Priestley Dean, College of Pharmacy ABSTRACT Nonactin is the parent compound of the macrotetrolide class of antibiotics, atypical cyclic polyethers, consisting of both enantiomers of nonactic acid subunits linked together via four ester linkages in a (+)(-)(+)(-) manner. The higher macrotetrolide homologs of nonactin are made up of successive substitutions of nonactic acid with homononactate or bishomononactate. The macrotetrolides act as ionophores, forming complexes with both monovalent and divalent ions. Organisms that produce antibiotics as a defense mechanism need to protect themselves from their own biosynthesis products. Self-protection is achieved using many methods such as modification of the antibiotic itself, modification of the cellular target, removal of the produced antibiotic to specific binding proteins, or changes in the cell wall. Many species employ several of these methods simultaneously, incorporating antibiotic modification steps into the biosynthetic pathway and using the other methods to offer layers of resistance. A genetic resistance element conferred nonactin resistance to a macrotetrolide sensitive host. The genetic element was cloned from the producer, Streptomyces griseus subsp. griseus. By sequence analysis the protein product, NonR, appeared to be an esterase. This work describes the subcloning of the gene nonR, its expression in a heterologous host, and examination of the activity of the expressed protein. Expression ii and purification the protein NonR, was accomplished using an affinity tag; the enzyme proved to be labile, losing activity throughout the purification process. NonR hydrolyzes the ester bonds of nonactin stereoselectively, cleaving between the acid of the (-)-nonactate and the alcohol of the (+)-nonactate. The order of hydrolysis is first the closed chain parent, nonactin, is cleaved to an open chain tetramer, followed by cleavage of the tetramer to two dimer species, Under conditions of high protein concentration, NonR will hydrolyze the dimer species to the monomer, nonactic acid. This hydrolysis causes nonactin to lose its biological activity. To meet our synthesis needs we set out to increase the yields of nonactin that is produced by fermentation. By a mix of strain selection, recipe and process improvement, nonactin production improved from 0.1 g·L-1 in stirred tank reactors to 4 g·L-1. iii ACKNOWLEDGMENTS I would like to acknowledge and thank my adviser Professor N. D. Priestley and all of my fellow co-workers at both The Ohio State University and at The University of Montana. iv VITA December 8, 1971…………………………………… …Born – Lancaster, California 1996……………………………………………………..B.S. Biology, B.A. Chemistry 1997-present…………………………………………….Graduate Teaching Associate and Graduate Research Associate, The Ohio State University FIELDS OF STUDY Major Field: Pharmacy v TABLE OF CONTENTS Abstract...........................................................................................................................ii Acknowledgments ..........................................................................................................iv Vita .................................................................................................................................v List of Tables..................................................................................................................ix List of Figures .................................................................................................................x Chapters 1. Introduction .............................................................................................................1 1.1 The importance of natural products .................................................................1 1.2 Fatty acid biosynthesis ....................................................................................5 1.3 Type I polyketide biosynthesis ........................................................................8 1.3.1 Macrolide biosynthesis .....................................................................10 1.3.2 Polyene biosynthesis .........................................................................12 1.3.3 Polyether biosynthesis ......................................................................14 1.4 Type II polyketide biosynthesis .....................................................................19 1.5 Type III polyketide biosynthesis ....................................................................23 1.6 Mechanisms of self resistance .......................................................................25 1.6.1 Antibiotic modification for self resistance.........................................29 1.6.1.1 N-acetylation and O-phosphorolation.................................32 1.6.1.2 O-glycosylation.................................................................34 1.6.1.3 Amino acid modification...................................................35 1.6.2 Resistance at the target site ..............................................................38 1.6.2.1 Resistance at the ribosome.................................................39 1.6.2.2 Non-ribosomal target sites.................................................41 1.6.2.2.1 DNA gyrase inhibitors.........................................42 1.6.2.2.2 Differential expression of EF-Tu factors ..............44 1.6.2.2.3 Glycedraldehyde-3-phosphate dehydrogenase ......46 1.6.3 Efflux pumps ....................................................................................47 1.6.3.1 ABC transporters...............................................................47 vi 1.6.3.2 Proton motive force efflux pumps ......................................49 1.6.4 Esterases that confer drug resistance .................................................50 2. History and importance of nonactin........................................................................51 2.1 Introduction to nonactin and the macrotetrolides ...........................................51 2.2 Isotope labeling experiments.........................................................................55 2.3 Biochemical studies.......................................................................................70 3. Characterization of an esterase that confers self resistance to nonactin ...................79 3.1 Introduction...................................................................................................79 3.2 In Silico studies of NonR...............................................................................82 3.3 Development of an ESI-MS assay .................................................................85 3.4 Examinaltion of S. griseus fermentation extracts by ESI-MS.........................86 3.5 Examination of the dependence of nonactin biosynthesis on ATP..................89 3.6 Analysis of NonL ........................................................................................105 3.7 Characterization of Non R and its products...................................................107 3.7.1 Cloning of nonR into an expression vector......................................107 3.7.2 Determination of NonR activity......................................................109 3.7.3 Scale up of NonR production for dimer isolation.............................114 3.7.4 NMR characterization of the dimer species.....................................115 3.7.5 Determination of NonR enantioselective cleavage of nonactin........115 3.7.6 Examination of the dimer species antibacterial activity ...................123 3.7.7 Purification of NonR.......................................................................129 3.7.8 Determination of NonR cofactor dependency..................................142 3.7.9 Determination of the optimal pH for NonR activity........................143 3.7.10 Probing the catalytic triad by point mutagenesis..............................143 3.8 Subcloning and expression of nonD.............................................................148 3.9 Discussion...................................................................................................149 4. Optimization of nonactin production in S. griseus ................................................152 4.1 Introduction.................................................................................................152 4.2 Assay for biomass and macrotetrolide concentration ...................................153 4.3 Fermentation media.....................................................................................154 4.4 Initial fermentation conditions .....................................................................154 4.5 Addition of oxygen to a fermentation batch .................................................155 4.6 Optimization of aeration conditions .............................................................158 4.7 Optimization of stir rate...............................................................................158 4.8 Optimization of temperature conditions .......................................................158
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