Investigations into the Structure and Function of Type I Polyketide Synthases by Aaron A. Koch A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Cancer Biology) in the University of Michigan 2017 Doctoral Committee: Professor David H. Sherman, Chair Professor Mark Day Professor Tom Kerppola Professor Benjamin Margolis Professor Janet Smith Aaron Andrew Koch [email protected] ORCID iD: 0000-0001-7488-9074 © Aaron Andrew Koch 2017 Acknowledgements My scientific journey has been as linear in trajectory as a sinusoidal wave function. I am forever indebted to a multitude of mentors who invested their time and energy into keeping my path towards a productive endpoint. During my undergraduate studies at Grand Valley State University I was blessed to work for mentors fully committed to the education and advancement of their students. I thank Professor David Leonard for instigating my fascination with biochemistry, especially enzymes. I am also grateful to Professor Richard Rediske for providing me the opportunity to enter the world of natural products research, a passion with which I am still afflicted today despite my initial assignment of collecting and analyzing raw sewage. During these years I was also fortunate to receive the opportunity to perform research at my future graduate school as a summer student in Dr. Colin Duckett’s lab. I thank professor Duckett for graciously providing me with this opportunity, scientific mentorship, and a paycheck; all of which were desperately needed. The inspiration and guidance of these mentors during my formative years ultimately led me to the laboratory of Dr. David Sherman at the University of Michigan where I have been fortunate to pursue my scientific interests in an exceptional research environment. I thank Dr. Sherman for affording me the opportunity to carry out the research outlined in this thesis under his guidance during my graduate studies. I also thank my committee members Dr. Mark Day, Dr. Tom Kerppola, Dr. Benjamin Margolis, and Dr. Janet Smith for providing insightful and encouraging feedback during the course of my time at the University of Michigan. Finally, I thank both former and present fellow scientists within the Sherman lab, especially Dr. Joe Chemler, Dr. Doug Hansen, Dr. Jeff Kittendorf, Dr. Andrew Lowell, Dr. Sean Newmister, and Vik Shende for their contributions and advice. I would be remiss if I did not make special mention of Dr. Hansen who served as my mentor during my graduate career on a volunteer basis (I hope that decision does not haunt you) and contributed significantly to many of the projects contained within this thesis. ii In addition to my professional and scientific guidance, I am immensely thankful for the support and encouragement of my friends and family that allowed me to successfully navigate this scientific labyrinth without full surrender of my sanity. I thank my parents Randy and Beth for fostering a loving and supportive environment conducive to exploration and curiosity during my childhood years. I am grateful to have had you as role models for treating others respectfully and living a life of personal accountability. Unless my future holds significant failures, in which case you will both receive full blame for my shortcomings. I also thank my siblings Jeremy and Laura Koch, Hannah and Zach Ricks, and Ben Koch; sharing life with you and your offspring has been an incredible experience and I am thankful for every one of you. Ben, I am especially grateful for you serving as both my brother and closest friend over the years which has greatly aided me in reaching this goal. I consider myself especially fortunate to have been welcomed into a second family during my time as a graduate student and I thank my parents-in-law Richard and Susan Wilson and sister-in-law Danielle Wilson for welcoming me into their family as one of your own and supporting me in so many ways. Finally, more than any other axially tilted rotations of the earth, I am thankful for two dates that occurred during my graduate studies that forever changed my life for the better. On 8-15-2015 I married my incredible wife Laura Jane Koch and words are inadequate to express my deep sense of gratitude for her unwavering love, support, and belief in me. Laura you have kept the wheels on the bus and I am blessed beyond belief to share life with you. Our son Everett Platte Koch entered the world on 4-8-2017, providing me with yet another source of gratitude and I am eagerly looking forward to the future with my family. iii Table of Contents Acknowledgements ..........................................................................................................ii List of Figures ..................................................................................................................vi List of Schemes .............................................................................................................. vii List of Tables ................................................................................................................. viii Abstract ...........................................................................................................................ix Chapter I: Introduction ..................................................................................................... 1 Therapeutic natural products ....................................................................................... 1 Polyketide natural products .......................................................................................... 3 Type I polyketide biosynthesis ..................................................................................... 4 The pikromycin biosynthetic pathway: a model system ............................................... 6 Engineering new polyketides ....................................................................................... 8 Thesis outline ............................................................................................................. 10 References ................................................................................................................. 13 Chapter II: Investigations into late stage PKS engineering ........................................... 16 Introduction ................................................................................................................ 17 Probing PikAIII-TE with unnatural pentaketides ......................................................... 18 Generation of hybrid TE PKS modules ...................................................................... 21 Role of the TE domain in engineered PKS modules .................................................. 23 Biocatalytic production of macrolactone analogs ....................................................... 29 Experimental procedures ........................................................................................... 31 References ................................................................................................................. 40 Chapter III: Interrogating thioesterase domains directly ................................................ 43 Introduction ................................................................................................................ 44 Stabilization of the Pik hexaketide ............................................................................. 45 Substrate controlled divergence in polyketide synthase catalysis .............................. 47 Confirmation of a thioesterase bottleneck in the processing of unnatural substrates . 53 iv A single active site mutation in the pikromycin thioesterase generates a more effective macrocyclization catalyst ........................................................................................... 54 PikAIII-TE with diastereomeric pentaketides ...................................................... 57 S148C Experimental procedures ........................................................................................... 61 References ................................................................................................................. 70 Chapter IV: Structural and mechanistic insights into type I PKS thioesterase catalysis 75 Introduction ................................................................................................................ 76 Trapping the TE acyl-enzyme intermediate ............................................................... 76 MD simulations .......................................................................................................... 84 Chemical lactonization ............................................................................................... 90 QM calculations ......................................................................................................... 92 Computational investigation of the Pik TE domain catalysis ...................................... 95 Engineering TE domains ............................................................................................ 96 Experimental procedures ........................................................................................... 98 References ............................................................................................................... 105 Chapter V: Discussion ................................................................................................. 108 Summary and insights ............................................................................................. 108 Future directions ...................................................................................................... 114 References ............................................................................................................... 119 v List of Figures Figure 1.1 Examples of clinically valuable bioactive natural products. ............................ 2 Figure 1.2 Type I polyketide synthase catalytic cycle ...................................................... 5 Figure 1.3 The pikromycin (Pik) biosynthetic pathway .................................................... 7 Figure 1.4 Laboratory methods for generating polyketide analogs .................................. 9 Figure 2.1 Proposed shunt pathways when macrocyclization is compromised ............. 21 Figure 2.3 Sequence alignment of the Pik, DEBS, and Juv thioesterase domains ....... 22 Figure 2.2 The thioesterase domains utilized in this study and the macrolactones they produce ......................................................................................................................... 22 Figure 2.4 Determination of the post-ACP restriction site for incorporating non-native TE domains ......................................................................................................................... 23 Figure 2.5 PAGE gel of hybrid TE PKS modules ........................................................... 24 Figure 2.6 LC-HRMS analysis of the purified product from reactions containing Juv Mod6-Pik TE with Tyl hexaketide 29 ............................................................................. 30 Figure 3.1 TE catalyzed macrolactonization or hydrolysis of an ACP-tethered polyketide intermediate .................................................................................................................. 44 Figure 3.2 Examples of previously studied native PKS chain elongation intermediates 46 Figure 3.3 Product distributions from previous in vitro analysis of PikAIV ..................... 48 Figure 3.4 A single active site mutation in the pikromycin thioesterase generates a more effective macrocyclization catalyst ................................................................................ 55 Figure 3.5 Incubation of diasteromeric pentaketides with PikAIII-TE ..................... 58 S148C Figure 4.1 Overall structure of the Pik TE dimer ........................................................... 77 Figure 4.2 General mechanism for trapping the TE acyl-enzyme intermediate through mutation of the catalytic triad ......................................................................................... 79 Figure 4.3 Expression and purification of MBP-Pik TE for substrate trapping S148CH268R studies ........................................................................................................................... 80 Figure 4.4 In vitro analysis confirms that Pik TE is catalytically inactive ......... 81 S148CH268R Figure 4.5 Pik TE is more amenable to in vitro analysis than TE ..... 82 S148CH268Q S148CH268R Figure 4.6 LC-HRMS analysis of Pik TE substrate labelling reactions ........... 83 S148CH268Q vi Figure 4.7 Acyl-enzyme starting structures for the MD simulations ............................... 84 Figure 4.8 Examination of the overall structural flexibility of the TE domain during the MD simulations .............................................................................................................. 85 Figure 4.9 Comparison of the reactive conformations for each acyl-enzyme intermediate obtained from clustering analysis of MD simulations with Pik TE .............................. 86 WT Figure 4.10 The TE-hexaketide contacts identified from the clustering analysis of the Pik TE MD simulations .................................................................................................... 88 WT Figure 4.11 Procatalytic sampling of Pik TE during MD simulations ............................. 89 Figure 4.12 Reaction coordinate diagram representing the relative free energies for Pik TE catalyzed macrolactonization of the native and C-11-epimerized hexaketides ........ 93 Figure 4.13 Lowest energy rate-limiting transition structures calculated with PCM/M06- 2X/6-31+G(d,p) for the abbreviated active site models ................................................. 94 Figure 4.14 DEBS TE possesses increased substrate flexibility ............................ 97 S139C vii List of Schemes Scheme 2.1 Evaluation of PikAIII-TE with a panel of truncated pentaketides ............... 19 Scheme 2.2 Reaction of PikAIII-TE with a panel of stereoisomer pentaketides ............ 20 Scheme 2.3 Reaction of Juv Mod6-Pik TE with Tyl hexaketide ..................................... 30 Scheme 3.1 Probing the Pik TE as an excised domain ................................................. 47 Scheme 3.2 Pik TE displays a high level of substrate stereospecificity ........................ 53 Scheme 3.3 Pik TE displays increased substrate flexibility .................................... 56 S148C Scheme 3.4 Reaction of PikAIII-TE with C-9-epimerized pentaketide ................... 59 S148C Scheme 3.5 Reaction of PikAIII-TE with native pentaketide .................................. 59 S148C Scheme 4.1 Yamaguchi macrolactonization of methyl protected hexaketides .............. 91 vii List of Tables Table 2.1 Yields from the Ni-NTA column purification of the hybrid TE PKS modules ... 25 Table 2.2 Screening of hybrid TE modules with Pik pentaketide ................................... 26 Table 2.3 Evaluation of TE hybrids with Pik hexaketide ................................................ 27 Table 2.4 Reaction of Juv Mod7 hybrids with Tyl hexaketide 29 ................................... 29 Table 3.1 Evaluation of stabilized Pik hexaketides with Pik TE ..................................... 49 Table 3.2 Evaluation of NBOM protected Pik hexaketides without photolysis ............... 50 Table 3.3 Evaluation of stabilized Pik hexaketides with PikAIV and MM-NAC extender unit ................................................................................................................................ 52 Table 3.4 Steady state kinetic values for Pik TE and TE . ................................... 56 WT S148C viii