University of South Carolina Scholar Commons Theses and Dissertations 2017 Elucidation Of Protein Interactions And The Re- Tuning Of Porphyrin Electronics For Hydroxylases Steven Charles Ratigan University of South Carolina Follow this and additional works at: https://scholarcommons.sc.edu/etd Part of the Chemistry Commons Recommended Citation Ratigan, S. C.(2017). Elucidation Of Protein Interactions And The Re-Tuning Of Porphyrin Electronics For Hydroxylases. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/4482 This Open Access Dissertation is brought to you by Scholar Commons. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. ELUCIDATION OF PROTEIN INTERACTIONS AND THE RE-TUNING OF PORPHYRIN ELECTRONICS FOR HYDROXYLASES by Steven Charles Ratigan Bachelor of Arts The Citadel, 2005 Bachelor of Science University of South Carolina, 2009 Master of Arts The Citadel, 2012 Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy in Chemistry College of Arts and Sciences University of South Carolina 2017 Accepted by: Thomas Makris, Major Professor F. Wayne Outten, Committee Member John Lavigne, Committee Member Zhengqing Fu, Committee Member Cheryl L. Addy, Vice Provost and Dean of the Graduate School © Copyright by Steven Charles Ratigan, 2017 All Rights Reserved. ii DEDICATION I dedicate this work to my family, who have always been there for me. Without their support, my time in academia would have been much more difficult. I would also like to thank Dr. David Donnell of The Citadel Dept. of Biology for teaching me everything I know about molecular biology, which proved to be a huge help early on in this program. iii ACKNOWLEDGEMENTS I would like to thank my mentor Dr. Makris for attempting to patiently deal with me for six years in this program. While I swore I would never be one of those people to sum things up with a generic quote, I’m going to do it anyway with “It was the best of times, it was the worst of times” - Charles Dickens I would also like to thank my fellow labmates. My early years were spent with two very interesting science personalities in Job Grant and Jason Hseih. Job nailed down a framework for analyzing high-valent intermediates in wild-type P450olet and Jason’s aminoacyl-CoA was the only batch anybody has been able to make since. Next came Jose Amaya who brought the chill to the lab and basically nailed down a framework for turnover analysis for my GC work. Courtney Wise, one of my former undergrads, followed Jose and dutifully wrote down, in excruciating detail, the procedures for doing anything in the lab. Finally, Olivia Manley arrived a little too late for knowledge to pass either way, but I think she’ll figure her stuff out. I would also like to thank Julia Bian, the only other undergrad that I mentored (besides Courtney) that it has been true honor to work with. iv ABSTRACT Non-Ribosomal Peptide Synthetases (NRPSs) and their exogenous tailoring partners have been heavily studied but not in the context of non-cognate systems. Orf78, a dinuclear iron β-hydroxylase from the lysobacter pathway and homologous to CmlA from the chloramphenicol pathway, is used to test affinities for one native and two non-native T-domains. Results indicate that there is enough difference between Type I and Type II NRPS systems to disfavor common recognition motifs. Additionally, the β-hydroxylase P450sky, from the skyllamycin biosynthetic pathway, is used in conjunction with the NRPS AT- domain NikP1AT from the nikkomycin biosynthetic pathway, in lieu of the homolog P450nikQ. The second portion of the thesis will discuss the creation of a sustainable source of biodiesel products are currently a goal of ‘green’ programs. The fatty acid decarboxylase P450olet uses the cheaply obtainable hydrogen peroxide as the oxidant to achieve a high-valent iron species. Substitution of the iron for manganese in the porphyrin scaffold raises the redox potential and forms a Mn(IV)-oxo complex that is too weak to perform C-H bond abstractions. Alternatively, substitution of the iron-protoporphyrin IX with iron- mesoporphyrin IX lowers the redox potential, increasing the amount of hydroxylated fatty acid at lower carbon chain lengths but also leads to a decrease in the efficiency in multiple turnover reactions. v TABLE OF CONTENTS DEDICATION ........................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................ iv ABSTRACT .............................................................................................................. v LIST OF TABLES ..................................................................................................... viii LIST OF FIGURES .................................................................................................... ix CHAPTER 1: A COMPARISON OF PCP DOMAINS AND THEIR RECOGNITION WITH A DINUCLEAR IRON HYDROXYLASE FROM THE LYSOBACTIN BIOSYNTHETIC PATHWAY .................................................................................................... 1 1.1 INTRODUCTION .......................................................................................... 1 1.2 MATERIALS AND METHODS ......................................................................... 6 1.3 RESULTS ................................................................................................. 13 1.4 DISCUSSION ............................................................................................ 22 1.5 CONCLUSION ........................................................................................... 25 1.6 REFERENCES .......................................................................................... 26 CHAPTER 2: DETERMINATION OF THE ROLE OF A NON-RIBOSOMAL PEPTIDE SYNTHETASE IN THE RECOGNITION OF A NON-COGNATE TAILORING ENZYME .. 41 2.1 INTRODUCTION ....................................................................................... 41 2.2 MATERIALS AND METHODS ...................................................................... 51 2.3 RESULTS ................................................................................................ 60 2.4 DISCUSSION ........................................................................................... 65 2.5 CONCLUSION ............................................................................................ 6 vi 2.6 REFERENCES ......................................................................................... 69 CHAPTER 3: THE SUBSTITUTION OF MANGANESE-PROTOPORPHYRIN IX INTO CYTOCHROME P450OLET ............................................................................ 93 3.1 INTRODUCTION ....................................................................................... 93 3.2 MATERIALS AND METHODS ...................................................................... 97 3.3 RESULTS AND DISCUSSION .................................................................... 104 3.4 CONCLUSION ........................................................................................ 115 3.5 REFERENCES ....................................................................................... 115 CHAPTER 4: RE-TUNING THE REACTIVITY OF THE FERRYL-OXO INTERMEDIATES IN P450OLET THROUGH SUBSTITUTION WITH IRON-MESOPORPHYRIN IX ........... 137 4.1 Introduction ........................................................................................ 137 4.2 Materials and Methods ....................................................................... 145 4.3 Results ............................................................................................... 151 4.4 Discussion .......................................................................................... 157 4.5 Conclusion ......................................................................................... 159 4.6 References ......................................................................................... 160 LIST OF TABLES Table 1.1 Sequence identity of 3 cognate and 2 non-cognate PCPs for P450sky ................................................................................................... 40 Table 3.1 Rates of Mn(IV)-oxo formation reacted with differing concentrations of peracetic acid ......................................................................................... 133 Table 4.1 Kinetic rates associated with PPolet and MPolet for Cpd. I and Cpd. II decay ........................................................................................... 177 Table 4.2 Turnover data for MPolet and PPolet ................................................ 177 LIST OF FIGURES Figure 1.1 Diagrams of the hydroxylation reaction with P450sky and the lysobactin antibiotic…………………………………………….………...…...34 Figure 1.2 Diagrams of Orf78 endogenous ligands and the active-site structure ................................................................................................... 34 Figure 1.3 Size-exclusion chromatography plot of Orf78 .................................... 35 Figure 1.4 Reductive titration of Orf78 ................................................................ 35 Figure 1.5 Optical spectrum of Orf78 incubated with sodium azide .................... 36 Figure 1.6 EPR spectra of mixed-valent Orf78, dithionite saturation, power saturation,