Spectroscopic and mechanistic investigation of the enzyme mercaptopropionic acid dioxygenase (MDO), the role of the conserved outer-sphere 'catalytic triad' in directing promiscuous substrate-specificity A DISSERTATION Presented to the faculty of the Graduate School of The University of Texas at Arlington in Partial fulfillment Of the requirements of the Degree of Doctor in Philosophy in CHEMISTRY by SINJINEE SARDAR Under the guidance of Dr. Brad S. Pierce 2nd May, 2018 ACKNOWLEDGEMENT It gives me immense pleasure to thank my compassionate guide Dr. Brad S. Pierce, for his exemplary guidance to accomplish this work. It has been an extraordinary pleasure and privilege to work under his guidance for the past few years. I express my sincere gratitude to my doctoral committee members Prof. Frederick. MacDonnell, Dr. Rasika Dias, and Dr. Jongyun Heo for their valuable advice and constant help. I would also like to thank the Department of Chemistry and Biochemistry of UTA for bestowing me the opportunity to have access to all the departmental facilities required for my work. Here, I express my heartiest gratitude to the ardent and humane guidance of my senior lab mates Dr. Josh Crowell and Dr. Bishnu Subedi which was indispensable. I am immeasurably thankful to my lab mates Wei, Mike, Phil, Nick, Jared and Sydney for their constant support and help. It has been a pleasure to working with all of you. The jokes and laughter we shared have reduced the hardships and stress of graduate school and had made the stressful journey fun. To end with, I utter my heartfelt gratefulness to my parents and my sister for their lifelong support and encouragement which helped me chase my dreams and aspirations and sincere appreciation towards my friends who are my extended family for their lively and spirited encouragement. 2nd April,2018 2 Abstract Spectroscopic and mechanistic investigation of the enzyme mercaptopropionic acid dioxygenase (MDO), the role of the conserved outer-sphere 'catalytic triad' in directing promiscuous substrate-specificity Sinjinee Sardar, Ph.D. The University of Texas, Arlington Supervising professor: Dr. Brad. S. Pierce Mercaptopropionate dioxygenase (MDO) from Aztobacter Vinelandii is a non-heme mononuclear iron enzyme that catalyzes the oxygen dependent conversion of 3-mercaptopropionic acid ( 3mpa ) to produce 3-sulfinopropinioic acid ( 3spa ). Nearly, all thiol dioxygenases have a conserved ‘catalytic triad ’ within the active site, consisting of three outer Fe-coordination sphere residues S155, H157 and Y159. With the notable exception of mammalian cysteamine (2- aminoethanethiol) dioxygenase (ADO), this ‘ catalytic triad ’ is universally conserved across phylogenic domains. X-ray crystallographic studies clearly show a hydrogen bond network connecting the ‘ catalytic triad ’ to the mononuclear iron site; however, its influence on substrate specificity and catalytic efficiency is poorly understood. In this work, kinetic and spectroscopic characterization of Av MDO is presented, focusing especially on the role of the conserved ‘catalytic triad’ on enzyme catalysis. In addition, pH/pD-dependent to solvent kinetic isotope (SKIE) experiments and proton inventory experiments were performed to investigate rate limiting proton- dependent steps during catalysis. Complementary viscosity studies are presented to probe diffusion limited steps in steady-state reactions. 3 Comparative studies were performed for reactions with L-cysteine and 3mpa as a substrate, for the active site variants, H157N and Y159F to investigate the role of specific residues within the ‘catalytic triad’. Taken together with supporting EPR and Mössbauer spectroscopic studies, a mechanistic model is proposed in which the ‘ catalytic triad ’ serves to gate proton delivery to the substrate-bound Fe-site. These studies suggest that the primary role of the ‘ catalytic triad ’ is to neutralize the negative charge of the substrate-carboxylate group in the rate-limiting non-chemical steps leading up to O 2-activation. 4 Table of contents Acknowledgements………………………………………………………………………….2 Abstract………………………………………………………………………………………3 List of illustrations…………………………………………………………………………. 9 List of tables……………………………………………………………………………….11 List of Schemes………………………………………………………………………………12 Chapter 1 Introduction to Mononuclear Non-heme iron enzymes………………………….14 Enzymes containing 2 His 1 carboxylate facial triad……………………………………… 16 Catechol Cleaving Dioxygenase…………………………………………………………....18 Extradiol dioxygenase……………………………………………………………………….19 Intradiol dioxygenase……………………………………………………………………….20 Rieske Dioxygenase…………………………………………………………………………21 α-Ketoglutarate dependent enzymes………………………………………………………. 25 Pterin-Dependent Hydroxylases……………………………………………………………28 Enzymes Biosynthesis of antibiotics……………………………………………………… 31 ACCO………………………………………………………………………………………32 Isopenicillin N synthase…………………………………………………………………… 34 Introduction of the 3-His facial triad……………………………………………………….36 5 Cysteine dioxygenase…………………………………………………………………………….38 3- Mercaptopropionic acid dioxygenase………………………………………………………...41 Chapter 2 “The ‘Gln-Type’ thiol dioxygenase from Azotobacter vinelandii is a 3- mercaptopropionic acid dioxygenase…………………………………………………………….44 Introduction………………………………………………………………………………………44 Materials and method……………………………………………………………………………47 Expression of Av MDO…………………………………………………………………………47 Enzyme purification…………………………………………………………………………….48 Enzyme assays………………………………………………………………………………….49 Synthesis of 3-sulfinopropionic acid……………………………………………………………50 Data analysis…………………………………………………………………………………….51 Anaerobic work…………………………………………………………………………………52 Physical methods……………………………………………………………………………….52 Nitric oxide additions……………………………………………………………………………53 Results………………………………………………………………………………………….54 Purification of Av MDO……………………………………………………………………….54. Steady- state kinetics of Av MDO catalyzed reactions…………………………………… ….58 pH effects………………………………………………………………………………………59 EPR spectroscopy of substrate bound Av MDO iron-nitrosyl active site Av ES-NO………….61 6 Discussion………………………………………………………………………………………68 Chapter 3 Solvent kinetic isotope effects on 3-Mercaptopropionic acid dioxygenase(MDO) from Azotobacter vinelandii reveals ionization events associated with substrate binding are rate limiting Introduction………………………………………………………………………………………78 Materials and methods…………………………………………………………………………...80 Expression and Purification and of Av MDO……………………………………………………82 Enzyme assays…………………………………………………………………………………...82 Synthesis of dianionic 3-sulfinopropionic acid……………………………………………………………83 Solvent kinetic Isotope effects and proton inventory……………………………………………84 Viscosity studies……………………………………………………………………………….84 Data analysis…………………………………………………………………………………….84 Results………………………………………………………………………………………….87 Solvent kinetic isotope effects………………………………………………………………….87 Proton inventories……………………………………………………………………………….91 Viscosity…………………………………………………………………………………………93 Discussion………………………………………………………………………………………94 Chapter 4: Thiol dioxygenase turnover yields benzothiazole products from 2-mercaptoaniline and O 2-dependent oxidation of primary alcohols. Introduction…………………………………………………………………………………100 7 Materials and Method………………………………………………………………………105 Enzyme purification…………………………………………………………………………106 Reagents and stock solutions……………………………………………………………….106 Synthesis of 2-phenybenzothiazole (2p-bt) …………………………………………………106 UV-visible experiments………………………………………………………………………107 Oxygen Electrode…………………………………………………………………………….108 HPLC sample preparation ……………………………………………………………………109 HPLC instrumentation and analysis………………………………………………………….109 LC-MS/MS (multiple reaction monitoring) ………………………………………………….109 GC-MS/MS sample preparation……………………………………………………………….110 GC-MS/MS Instrumentation and Method…………………………………………………….110 Results and Discussion……………………………………………………………………….110 References……………………………………………………………………………………123 Biographical information…………………………………………………………………….144 8 List of Illustrations Figure 1-1: 2-His-1-carboxylate facial triad ………………………………………….……….16 Figure 1-2: Active site structure of Intradiol and Extradiol dioxygenase enzyme………….….19 Figure 1-3: Active site structure of a arene cis-hydroxylating oxygenase…………………….23 Figure 1-4: General structure of α-ketoglutarate cofactor…………………………………….26 Figure 1-5: Structure of the Pterin co-factor(tetrahydrobiopterin)…………………………….28 Figure 1-6: Crystal structure Pseudomonas aeruginosa ‘Gln-type’ MDO…………………….31 Figure 2-1. Crystal structure of the substrate-bound R. norvegicus CDO active site………….43 Figure 2-2. Steady-state kinetics of Av MDO-catalyzed 3spa and csa formation …………….55 Figure 2-3. pH dependence of kcat and kcat /KM for Av MDO-catalyzed reactions with 3mpa , cys and ca ………………………………………………………………………………………….59 17 3 Figure 2-4. X-Band EPR spectra of Av ES -NO (S = /2) species……………………64 17 3 Figure 2-5. X-Band EPR spectra of Av ES -NO (S = /2) species produced using l-cysteine (cys ), ethanethiol ( et ), and cysteamine ( ca ) ………………………………….............66 Figure 3- 1: Crystal structure of the substrate-bound ‘Arg-type’ Bacillus subtilis active site….78 Figure 3-2: pL-dependence of steady-state kinetic parameters of MDO using 3 mercaptopropionic acid and L-cysteine………………………………………………………88 Figure.3-3: Proton inventory of MDO catalyzed formation of product at pL 6.0 and 8.2…………………………………………………………………………………………….91 9 Figure. 3-4: Effect of solvent viscosity on the maximal rate ( v0/[E]) of MDO catalyzed 3spa and csa formation……………………………………………………………………………………94 Figure 4-1. Crystal structure of the Rattus