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Development of a Four-Step Semi-Biosynthesis of the Anticancer Drug Paclitaxel and Its Analogues

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Development of a Four-Step Semi-Biosynthesis of the Anticancer Drug Paclitaxel and Its Analogues DEVELOPMENT OF A FOUR-STEP SEMI-BIOSYNTHESIS OF THE ANTICANCER DRUG PACLITAXEL AND ITS ANALOGUES By Chelsea Thornburg A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology ‒ Doctor of Philosophy 2015 ABSTRACT DEVELOPMENT OF A FOUR-STEP SEMI-BIOSYNTHESIS OF THE ANTICANCER DRUG PACLITAXEL AND ITS ANALOGUES By Chelsea Thornburg Paclitaxel (Taxol®) is a widely used chemotherapeutic drug with additional medical applications in drug-eluting stents as an anti-restenosis treatment. Paclitaxel is a structurally complex natural product with an excellent scaffold for designing analogs with pharmacological properties. To date, clinically approved analogs include docetaxel and cabazitaxel for the treatment of additional cancers. Currently, plant cell fermentation methods produce paclitaxel and large quantities of the precursors 10-deacetylbaccatin III (10-DAB) and baccatin III. The complexity of the semi-characterized ~19-step paclitaxel biosynthetic pathway limits bioengineering attempts. However, the availability of 10-DAB and baccatin III suggests a semi-biosynthetic pathway to paclitaxel starting with these precursors is feasible. We have designed a short, simple biosynthetic pathway, capable of making paclitaxel, analogs, and/or valuable precursors for the semi-synthesis of additional analogs of biological interest. The paclitaxel biosynthesis enzyme baccatin III: 3-amino-13-O-phenylpropanoyl CoA transferase (BAPT) and the bacterial (2R,3S)-phenylisoserinyl CoA ligase (PheAT) produce N-debenzoylpaclitaxel, N-debenzoyldocetaxel, or precursor analogs. The addition of the paclitaxel biosynthetic N-debenzoyltaxol-N-benzoyltransferase (NDTNBT) and the bacterial benzoate CoA ligase (BadA) produce paclitaxel or other N-acylated analogs. In this dissertation, BAPT and BadA are kinetically characterized. The substrate specificity of BadA was systematically investigated with a series of 24 substrates. Six crystal structures of BadA in complex with different substrates, including benzoyl AMP, are used to explain BadA reactivity and propose rational mutations (A227A, H333A, and I334A) that expand substrate specificity and provide insight into the BadA mechanism and connect with established acetylation regulatory mechanisms in bacteria. Major hurdles including solubility and substrate availability, were overcome in order to characterize BAPT activity in the proposed semi-biosynthetic pathway. BAPT was purified as a fusion protein with maltose binding protein and its (2R,3S)-phenylisoserinyl CoA substrate was biosynthesized. To our knowledge this is the first time (2R,3S)- phenylisoserinyl CoA has been isolated in quantitative yields high enough to allow for characterization of the Michaelis-Menten kinetic constants (kcat and KM) for BAPT. This dissertation also describes the combination of BAPT and a bacterial ligase (PheAT) to produce N-debenzoylpaclitaxel and N-debenzoyl-10-deacetylpaclitaxel, precursors of paclitaxel and docetaxel, respectively. Biosynthesis of a biologically active paclitaxel analog, N-2-furanyl-N-debenzoylpaclitaxel, using the aforementioned enzymes, is also demonstrated as proof-of-principle that this semi-biosynthetic pathway may shorten the number of steps required to make certain paclitaxel (and docetaxel) analogs of interest. Copyright by CHELSEA THORNBURG 2015 ACKNOWLEDGEMENTS I would like to acknowledge my advisor, Dr. Kevin Walker, for his support during my time here at Michigan State University. I also would like to acknowledge Dr. Dan Jones for all his advice and assistance in learning mass spectrometry. Dr. Jim Geiger kindly trusted me with his chromatography equipment and was a great collaborator for the BadA ligase crystallography work. The biochemistry and molecular biology (BMB) department has been a wonderful academic home. Faculty members were always willing to discuss any research problems I encountered along the way. The following professors sat down with me at some point and said helpful things: Bill Henry, Tom Sharkey, Honggao Yan, Kaillathe “Pappan” Padmanabhan, Charlie Hoogstraten, and Kristin Parent “KP”. I have to thank my family for all their love and support. My mom- Kristen Thornburg, my Papi- Mark Santas, my sisters- Caitlin Thornburg and Rhoda Brew-Appiah, my bro-in-law Matt Seidel, and my niece Madison are always there for me even though they have no idea what I do all day. My GREAT aunt Frankie and uncle Jim welcomed me into their home and are two of my favorite people. I am also grateful to the lovely Janelle and James Sabo (and the girls: Claire, Katherine, and Sophia) for welcoming me into their home for Thanksgiving these past few years. I also need to thank all the people I have lost during my doctoral program. My grandfather- Newton Thornburg, my grandmother- Cloteel Atkins, my dear friend Pam Movalson and her daughter, Christine. I miss you all. v TABLE OF CONTENTS LIST OF TABLES ...............................................................................................................x LIST OF FIGURES ........................................................................................................... xi KEY TO ABBREVIATIONS ......................................................................................... xvii Chapter 1. Clinical use and production of paclitaxel and analogs of clinical interest .........1 1.1 Introduction ...........................................................................................................1 1.1.1 Clinical uses of paclitaxel ............................................................................. 1 1.1.2 Clinical uses of paclitaxel analogs ................................................................ 3 1.1.3 A brief history of paclitaxel .......................................................................... 3 1.1.4 Paclitaxel mode of action .............................................................................. 5 1.1.5 Paclitaxel biosynthesis .................................................................................. 7 1.1.6 Paclitaxel production .................................................................................. 11 1.1.7 Semi-biosynthesis of paclitaxel, precursors, and analogs ........................... 14 REFERENCES ...............................................................................................................19 Chapter 2. Kinetically- and crystallographically-guided mutations of a benzoate CoA ligase (BadA) elucidate mechanism and expand substrate permissivity ...........................33 2.1 Introduction .........................................................................................................33 2.2 Experimental .......................................................................................................38 2.2.1 Materials ..................................................................................................... 38 2.2.2 Plasmids ...................................................................................................... 39 2.2.3 BadA protein expression and purification .................................................. 39 2.2.4 BadA kinetic assays .................................................................................... 40 2.2.5 BadA assay analysis by liquid chromatography mass spectrometry .......... 41 2.2.6 BadA mutations .......................................................................................... 42 2.2.7 Kinetic analysis ........................................................................................... 43 2.2.8 BadA crystal structures ............................................................................... 43 2.2.8.1 Crystallization of R. palustris benzoate: coenzyme A ligase (BadA) ......43 2.2.8.2 Co-crystallization to obtain the ligand bound structure ...........................44 2.2.8.3 Data processing and refinement of BadA ................................................44 2.2.9 Calculation of covalent van der Waals volumes and lengths ..................... 45 2.3 Results .................................................................................................................46 2.3.1 Solving the BadA structure ......................................................................... 46 2.3.1.1 Domain orientation ...................................................................................46 2.3.1.2 Features of the BadA active site ...............................................................47 2.3.2 Kinetic properties of BadA ......................................................................... 50 2.3.3 Substrate turnover by BadA ........................................................................ 50 2.3.3.1 Halogenated benzoates .............................................................................50 2.3.3.2 Benzoates with strongly electron-withdrawing substituents ....................52 2.3.3.3 Benzoates with strongly electron-donating substituents ..........................52 vi 2.3.3.4 Turnover of heteroaromatic carboxylates ................................................53 2.3.3.5 Turnover of non-aromatic carbocycle carboxylates .................................54 2.3.4 Rational Mutation of the BadA Active Site ................................................ 54 2.3.4.1 Ala227Gly-BadA mutant .........................................................................56 2.3.4.2 Ile334Ala-BadA mutant ...........................................................................56
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