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University of Cincinnati UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of: in: It is entitled: This work and its defense approved by: Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________ Functional Genomics of Xenobiotic Detoxifying Fungal Cytochrome P450 system A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati In partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) In the Department of Environmental Health of the College of Medicine March 2008 by Venkataramanan Subramanian M.S., University of Southern Mississippi, 2002 M.Sc., University of Mumbai, India, 1997 B.Sc., University of Mumbai, India, 1995 Committee Chair: Jagjit. S. Yadav, Ph.D. Associate Professor Department of Environmental Health University of Cincinnati Abstract The white rot fungus Phanerochaete chrysosporium is primarily known for its ability to degrade a wide range of xenobiotic compounds including the highly recalcitrant polycyclic aromatic hydrocarbons. The natural substrate of this basidiomycete fungus is however, lignin, the most abundant aromatic polymer on earth. The versatililty of this fungus in breaking down a wide array of compounds arises from the presence of a highly nonspecific enzyme system (peroxidase enzyme system) in its repertoire. Most of the research involving degradation of toxic chemicals has focused on this biodegrading enzyme machinery. Cytochrome P450 monooxygenases (P450s) on the other hand, are heme-thiolate proteins that are known to be involved in metabolism of endogenous compounds as well as xenobiotic compounds in higher eukaryotes. Nearly 150 P450s are present in this organism, which is the highest number known till date among fungal species. Based on the sequence similarity criteria and our phylogenetic analysis, these P450s have been classified under 12 families and 23 sub-families. Despite indirect evidences suggesting the role of P450s in oxidation of xenobiotics, there have been hardly any reports on characterization and role of individual P450s either in regulation of physiological processes or in direct metabolism of xenobiotics in this organism. Here we characterized and investigated the role of P450 enzymes in two different mechanisms in this fungus. One, indirect involvement of P450s in peroxidase–mediated oxidation of xenobiotics, and two, direct involvement of P450s in metabolism of xenobiotics. In order to achieve the first objective, we investigated the role of PC-bph gene, the only member of the P450 CYP53 in synthesis of a secondary metabolite, veratryl alcohol, which regulates the activity of the peroxidase enzyme system of this fungus. In order to achieve the second objective, we used the functional genomic approach based on a custom-designed microarray and heterologous expression of the iii components of the P450 enzyme system (P450 and its associated electron transfer proteins) in this white rot fungus. iv v Acknowledgements First and foremost I offer my sincerest gratitude to my graduate advisor, Dr. Jagjit Yadav for his supervision, advice and guidance since day one of my research in his laboratory. He has always been a great motivating factor providing me with unflinching encouragement and support for all these years. Being in his laboratory has provided me with the extraordinary scientific exposure that has inspired me and enriched my growth as a student, researcher and a scientist want to be. My heartfelt thanks to each of my committee members, Drs. David Askew, Brian Kinkle, Jodi Shann, and Howard Shertzer for their constructive suggestions and criticisms that has resulted in the generation of this thesis. They have always been available for me whenever I needed them for any technical advice. I gratefully acknowledge Dr. David Warshawsky for initially being part of my thesis committee. Many thanks to Dr. Askew for providing me with the necessary resources to work on the Aspergillus niger transformation in his laboratory. I also thank Mike Miley for imparting me a hands-on training in Dr. Askew’s laboratory. Collective and individual acknowledgements are also owed to my lab colleagues for their constant support and encouragement extended to me for all these years. My special thanks to Harshavardhan Doddapaneni for being a great friend and colleague in the laboratory, for involving me in many of his projects, and for always being available with his valuable scientific advice. Many thanks to Izhar Khan, Suresh Babu Selvaraju, Renuka Kapoor, Manish Gupta, Hansraj Bangar, Huiyan Wang, Eunice Varughese, and Fannie Papatoli for creating such a pleasant atmosphere to work in the lab and for sharing both the good and bad times together during my tenure. I convey special acknowledgements to Dr. George Smulian and Dr. Francisco Gomez for training me to use the Maldi-TOF instrument in their laboratory, as well as providing the Pichia and the Agrobacterium strains used in my thesis. I also thank Dr. Rajiv Soman for training me to use the Mass spectrometry instrument in his laboratory. vi It is a pleasure to pay tribute to the faculty and staff of the first floor, who always kept their doors open for me and helped me with both scientific and personal issues in every possible way. I was constantly helped by all the faculty, staff, and students of this division in one way or the other. It is also my pleasure to mention the names of Scott Schneider and Saikumar Karyala for being such wonderful friends and for giving me a cherishable time and for accompanying me to the Indian restaurants. I will definitely miss their companionship. On a personal front, I would like to thank my ‘Cinci gang’ - Ritesh, Prodipto, Anu, Anup, Rohit, Chris, Bikram, Renuka, Ramya, Subbu and many others, for their everlasting friendship, entertaining company, and thoughtful support especially during those days when I needed them the most. What would I do without my family? My parents deserve special applause for their perpetual support and prayers. I offer my deepest gratitude to my father Late K.N. Subramanian and my mother Brahada Subramanian for their blessings and prolonged encouragement that has motivated me to achieve my goal as what I am today. Words fail me to express my appreciation to my brother S.L. Narayan who has been an indispensable moral support for me thereby strengthening my confidence especially after my father’s demise. Last but not the least, I owe a big “thank you” to my dear wife Mathangi whose love, patience and persistent confidence in me has taken the load off my shoulder towards the completion of my degree. vii Table of Contents List of tables……………………………………………………………………………………...11 List of figures.……………………………………………………………………………………12 Chapter I Introduction to the white-rot fungus, Phanerochaete chrysosporium and its biodegradative enzyme systems Introduction……………………………………………………………………………....16 Peroxidase enzyme system……….………………….…………………………………..22 Cytochrome P450 enzyme system.………………………………………………………23 Chapter II Proposed direct and indirect involvement of P450s in detoxification of xenobiotics Proposed role of P450s in peroxidase-mediated oxidation of xenobiotics (PAHs)..…….28 Proposed role of P450s in direct oxidation of xenobiotic compounds (PAHs)…….........32 Hypothesis………………………………………………………………………………..38 Chapter III Role of P450s in regulation of peroxidase-mediated degradation of xenobiotics via veratryl alcohol synthesis Introduction………………………………………………………………………………39 Materials and Methods…………………………………………………………………...40 Results…………………………..………………………………………………………..54 Discussion………………………………………………………………………………..65 Chapter IV Regulation and heterologous expression of p450 enzyme system components of the white rot fungus Phanerochaete chrysosporium Abstract…………………………………………………………………………………..73 viii Introduction………………………………………………………………………………75 Materials and Methods…………………………………………………………...............76 Results and discussion…………………………………………………………...............80 Conclusions………………………………………………………………………………90 References………………………………………………………………………………..92 Chapter V P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and purification of the expressed proteins Abstract…………………………………………………………………………………104 Introduction……………………………………………………………………………..105 Materials and Methods………………………………………………………………….107 Results…………………………………………………………………………………..114 Discussion………………………………………………………………………………118 References………………………………………………………………………………125 Chapter VI Role of P450 monooxygenases in degradation of the endocrine disrupting chemical nonylphenol by the white rot fungus Phanerochaete chrysosporium: biochemical and functional genomic evidences Summary………………………………………………………………………………..141 Introduction……………………………………………………………………………..142 Results…………………………………………………………………………………..144 Discussion………………………………………………………………………………148 Experimental Procedures……………………………………………………………….154 References………………………………………………………………………………159 ix Chapter VII Heterologous expression and purification of white rot fungal cytochrome P450 monooxygenase PC-2, a PAH-inducible P450 Introduction……………………………………………………………………………..173 Materials and Methods………………………………………………………………….174 Results…………………………………………………………………………………..179 Discussion………………………………………………………………………………181 Chapter VIII Conclusions……………………..………………………………………………………203
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