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Mechanistic Studies on Aldehyde Decarbonylase from Cyanobacteria: a New Enzyme for Alkane Biosynthesis

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Mechanistic Studies on Aldehyde Decarbonylase from Cyanobacteria: a New Enzyme for Alkane Biosynthesis Mechanistic Studies on Aldehyde Decarbonylase from Cyanobacteria: A New Enzyme for Alkane Biosynthesis by Debasis Das A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in The University of Michigan 2014 Doctoral Committee: Professor E. Neil G. Marsh, Chair Professor Carol A. Fierke Associate Professor Nicolai Lehnert Associate Professor Bruce A. Palfey Professor Stephen W. Ragsdale @Debasis Das 2014 Dedication To my parents _ for their love, support and encouragement ii Acknowledgements I would like to take this opportunity to convey my deep gratitude to my research advisor, Prof. Neil Marsh, for his endless patience, encouragements, guidance and valuable suggestions throughout the design and accomplishment of the research described in this thesis. I would also like to express my great appreciation to Prof. Stephen Ragsdale and Prof. Carol Fierke for being extremely supportive with their guidance and for letting me use their lab facilities. I also thank Prof. Bruce Palfey, Prof. Nicolai Lehnert and Prof. Vincent Pecoraro for their valuable suggestions and guidance on my research. I would like to convey my special thanks to former Marsh lab members Dr. Dustin Patterson and Dr. Bekir Eser for being my mentors and teaching me all the necessary skills required for doing research in a biochemistry lab. I also thank the present and past Marsh group members Dr. Ben Buer, Dr. Bishwajit Paul, Dr. Fengming Lin, Dr. Yuta Suzuki, Dr. Jaehong Han, Dr. Roberto de la Saud Bea, Dr. Lei Li, Gabriel Roman, Matt Waugh, Ben Ellington, Matthew Demars, Tad Ogorzalek, Aaron Sciore, McKenna Schroeder, Dorota Marchael, Ben Levin and Umair Daimee for their productive collaborations, suggestions, friendship and for maintaining a fun-full atmosphere in the lab. I would like to extend my special thanks to Dr. Amelie Bernerd for her expert advices and help on expression of membrane proteins, Jim Windak for his great assistance with mass spectroscopy, Dr. Venky Basrur for his expertise and guidance on ES-MS/MS spectroscopy and Dr. Naresh Theddu for his valuable support on organic synthesis. I wish to acknowledge the encouragements provided by iii all my Ann Arbor friends especially Dr. Bishwajit Paul, Dr. Pralay Mitra, Dr. Susanta Ghosh, Dr. Trisha Sain and Dr. Saumen Chakraborty, Krishna and Riddhiman. iv Table of Contents Dedication ........................................................................................................................................ ii Acknowledgements ......................................................................................................................... iii List of Figures ................................................................................................................................ viii List of Appendices ......................................................................................................................... xvi List of Abbreviations .................................................................................................................... xvii Abstract ...................................................................................................................................... xix Chapter 1 Introduction ................................................................................................................... 1 1.1 Biofuels ........................................................................................................................... 1 1.1.1 Requirement for Biofuels .......................................................................................... 1 1.1.2 Different Types of Biofuels ....................................................................................... 2 1.1.3 Challenges Associated with Biofuel Production ....................................................... 4 1.2 Biosynthetic Pathways for Biofuels ................................................................................ 5 1.2.1 Alcohol-Derived Biofuels ........................................................................................... 5 1.2.2 Isoprenoid-Derived Biofuels ..................................................................................... 7 1.2.3 Fatty Acid-Derived Biofuels ....................................................................................... 8 1.3 Chemical Decarbonylation Reactions .......................................................................... 10 1.4 Biological Decarbonylation Reactions .......................................................................... 13 1.4.1 Acyl-CoA Reductase ................................................................................................ 14 1.4.2 Aldehyde Decarbonylase ........................................................................................ 17 1.5 Aims of This Work ........................................................................................................ 22 1.6 References .................................................................................................................... 25 Chapter 2 Conversion of Aldehydes to Alkanes by Cyanobacterial Aldehyde Decarbonylase .............................................................................................................. 29 2.1 Introduction ................................................................................................................. 29 v 2.2 Materials and Methods ................................................................................................ 32 2.3 Results and Discussion ................................................................................................. 42 2.3.1 Characterization and Properties of cAD ................................................................. 42 2.3.2 Activity and Kinetics of cAD .................................................................................... 46 2.3.3 Co-Product of cAD-Catalyzed Reaction ................................................................... 56 2.3.4 Nature of the Aldehyde Hydrogen and the New Hydrogen in Product Alkane. .................................................................................................................... 60 2.3.5 Shorter Chain Aldehydes as Substrate of cAD: Kinetics with Heptanal .................. 63 2.3.6 α,β -Unsaturated Aldehydes as Reversible Competitive Inhibitors of cAD ............ 66 2.3.7 Role of the Reducing System in cAD-Catalyzed Reaction ....................................... 71 2.3.8 Aldehyde Decarbonylases from Different Classes of Cyanobacteria ..................... 76 2.3.9 Oxygen Dependence of cAD-Catalytic Activity ....................................................... 78 2.4 Conclusions .................................................................................................................. 80 2.5 References .................................................................................................................... 82 Chapter 3 Investigations of Ferredoxin-Dependent Cyanobacterial Aldehyde Decarbonylase Activity ................................................................................................. 85 3.1 Introduction ................................................................................................................. 85 3.2 Materials and Methods ................................................................................................ 89 3.3 Results and Discussion ................................................................................................. 94 3.3.1 Characterization of Np cAD ..................................................................................... 94 3.3.2 Characterization of Ferredoxin ............................................................................... 94 3.3.3 Activity Assays of cAD in Presence of Ferredoxin ................................................... 95 3.3.4 Effect of Salt Concentrations on the Fd-Dependent Activity of cAD .................... 100 3.3.5 Comparison of Activity of Different cADs with Ferredoxins and PMS/NADH ...... 107 3.3.6 Evidences of Electron Transfer from Reduced Fd to cAD ..................................... 109 3.3.7 Cross-Linking of cAD with Fd................................................................................. 115 3.4 Conclusions ................................................................................................................ 118 3.5 References .................................................................................................................. 119 Chapter 4 Probing the Mechanism of Cyanobacterial Aldehyde Decarbonylase using a Cyclopropyl Aldehyde ................................................................................................ 122 vi 4.1 Introduction ............................................................................................................... 122 4.2 Materials and Methods .............................................................................................. 124 4.3 Results and Discussion ............................................................................................... 129 4.3.1 EPR Experiments to Explore the Interaction of cAD with Substrate Aldehyde ............................................................................................................... 129 4.3.2 Cyclopropyl Analog of Octadecanal as a Substrate of cAD ................................... 131 4.3.3 Covalent Modification of cAD
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