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Structural and Biochemical Insights Into Catalytic STRUCTURAL AND BIOCHEMICAL INSIGHTS INTO CATALYTIC MECHANISMS OF CAROTENOID CLEAVAGE OXYGENASES By XUEWU SUI Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Advisers: Drs. Philip D. Kiser and Krzysztof Palczewski Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY January 2017 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Xuewu Sui Candidate for the Doctor of Philosophy degree *. Philip Kiser (Thesis Advisor) Krzysztof Palczewski (Thesis Advisor) Jason Mears, PhD (Committee Chair) Johannes von Lintig, PhD (Committee Member) Vivien Yee, PhD (Committee Member) Matthias Buck, PhD (Committee Member) Date of Defense 09/02/2016 * We also certify that written approval has been obtained for any proprietary material contained therein. ii I dedicated this work to: My mentors and friends for inspiration, encouragement and help & My family for love and support iii Table of Contents Table of Contents List of Tables ......................................................................................................................x List of Figures ...................................................................................................................xi ACKNOWLEDGEMENTS ............................................................................................. xiv LIST OF ABBREVIATIONS .......................................................................................... xvi ABSTRACT ....................................................................................................................... 1 CHAPTER 1: INTRODUCTION AND BACKGROUND ON CAROTENOIDS AND CAROTENOID CLEAVAGE OXYGENASES .............................................................. 3 1.1 Introduction to carotenoids ................................................................................... 4 1.2 Introduction to carotenoid cleavage oxygenases ............................................. 5 1.3 Structural basis for carotenoid cleavage by CCOs........................................... 6 1.3.1 CCOs display high substrate and cleavage site specificities ................... 6 1.3.2 Conserved 3D architecture of CCOs ........................................................... 9 1.3.3 Structural features in the catalytic center .................................................. 10 1.3.4 A hydrophobic patch for CCOs membrane penetration.......................... 13 1.3.5 Substrate binding pocket in CCOs ............................................................. 16 1.4 Proposed CCOs reaction mechanisms ............................................................ 18 1.5 CCOs and human health .................................................................................... 20 1.6 Unresolved questions in the CCO field ............................................................ 22 iv 1.7 Goals, experiment outline and rationale for the project ................................. 23 Figures.......................................................................................................................... 26 CHAPTER 2: ANALYSIS OF CAROTENOID ISOMERASE ACTIVITY IN A PROTOTYPICAL CAROTENOID CLEAVAGE ENZYME, APOCAROTENOID OXYGENASE ................................................................................................................. 42 2.1 Introduction and background ............................................................................. 43 2.2 Experimental procedures.................................................................................... 46 2.2.1 Protein expression and purification ............................................................ 46 2.2.2 Protein Crystallization................................................................................... 47 2.2.3 Enzymatic assay and high-performance liquid chromatography (HPLC) analysis ..................................................................................................................... 48 2.2.4 Purification and mass spectrometric analysis of apocarotenoid product ................................................................................................................................... 50 2.2.5 Raman spectroscopy .................................................................................... 50 2.2.6 X-ray data collection, structure determination, refinement and analysis ................................................................................................................................... 51 2.3 Results ................................................................................................................... 52 2.3.1 ACO purification and enzymatic analysis.................................................. 52 2.3.2 HPLC analysis of the cleavage products .................................................. 54 2.3.3 In situ analysis of ACO reaction products by Raman spectroscopy ..... 55 2.3.4 Detergents and PEG affect ACO activity .................................................. 57 v 2.3.5 Structure of native ACO in the absence of substrate .............................. 58 2.3.6 ACO structure in Triton X-100..................................................................... 59 2.4 Discussion ............................................................................................................. 61 Tables ........................................................................................................................... 66 Figures.......................................................................................................................... 70 CHAPTER3: KEY RESIDUES FOR CATALYTIC FUNCTION AND METAL COORDINATION IN APOCAROTENOID CLEAVAGE OXYGENASE (ACO) ..... 88 3.1 Introduction and background ............................................................................. 89 3.2 Materials and Methods........................................................................................ 93 3.2.1 In silico ligand docking ................................................................................. 93 3.2.2 Molecular biology, protein expression and purification ........................... 93 3.2.3 Enzymatic assays, high performance liquid chromatography (HPLC), and mass spectrometry (MS) analyses ............................................................... 94 3.2.4 Determination of kinetic parameters .......................................................... 95 3.2.5 Protein crystallization, structural determination, and analysis ............... 96 3.3 Results ................................................................................................................... 97 3.3.1 Identification of potential substrate-interacting residues for mutagenesis studies....................................................................................................................... 97 3.3.2 ACO active site mutants impair catalytic activity to various degrees without altering regioselectivity ............................................................................. 98 vi 3.3.3 Active site mutants primarily affect maximal enzymatic activity rather than the Michaelis constant .................................................................................100 3.3.4 The crystal structure of W149A ACO reveals major disruptions in the substrate-binding cleft and metal coordination.................................................101 3.3.5 E150 in the second sphere is critical for metal binding, maintenance of active site structure and catalytic activity ..........................................................103 3.4 Discussion ...........................................................................................................105 Tables .........................................................................................................................111 Figures........................................................................................................................117 CHAPTER4: UTILIZATION OF DIOXYGEN BY CAROTENOID CLEAVAGE OXYGENASES .............................................................................................................129 4.1 Introduction and background ...........................................................................130 4.2 Materials and methods......................................................................................133 4.2.1 Phylogeny inference ...................................................................................133 4.2.2 Protein expression and purification ..........................................................134 4.2.3 Enzymatic assays and rate determination for background oxygen exchange ................................................................................................................135 4.2.4 Isotope labeling study in H218O .................................................................137 4.2.5 Sample deoxygenation and isotope labeling study in 18O2 ..................137 4.2.6 HPLC and mass spectrometric analyses of cleavage products ..........139 4.2.7 RPE microsome deoxygenation and isomerization activity assays ....139 vii 4.2.8 Protein crystallization, structural determination and analysis ..............140 4.3 Results .................................................................................................................141 4.3.1 Assessment of apocarotenoid solvent back-exchange.........................141 4.3.2 Apocarotenoid labeling studies in the presence of
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