AN ABSTRACT OF THE THESIS OF Mei-Fei Yueh for the degree of Doctor of Philosophy in Toxicologypresented on January 4, 1999. Title: Identification and Characterization ofFlavin-Containing Monooxygenase Isoform 2 (FMO2) in Rhesus Monkey and Examination of anHuman FMO2 Polymorphism. Redacted for privacy Abstract Approved: David E. Williams Flavin-containing monooxygenase (FMO, EC1.14.13.8)comprises a family of xenobiotic-metabolizing enzymes that catalyze the oxygenation of awide variety of xenobiotics, most commonly nitrogen and sulfur. Mammals expressfive different FMOs in a species- and tissue- specific manner. FMO2, is expressedpredominantly in lung and differs from other FMOs in that it can catalyze the N-oxygenation ofcertain primary alkylamines. From its initial discovery as an unique form of FMO inlung, FMO2 has been studied primarily using a rabbit animal model. The initial goal of thisresearch was to characterize this protein in a primate animal model. To understand FMO2protein structure at the molecular level, we first cloned cDNAfrom a monkey lung cDNA library. Monkey FMO2 is expressed predominantly in lung. The high expressionlevels and broad substrate specificity in monkey, suggests that FMO2 plays a role in xenobioticmetabolism in this primate model. We then established a heterologous expression system to generate FMO2 with biological functionality in vitro. FMO2 from baculovirus-mediatedexpression resembled monkey and rabbit microsomal FMO2 immunochemically andcatalytically. The successful FMO2 expression in the baculovirus system will serve as a validtool for structure studies of protein functional domains, as well as, theamino acids responsible for enzyme properties of chimeras. Human FMO2encodes a truncated protein containing 471 amino acid residues, 64 amino acids shorter in its C-terminal thanorthologs in other species. We characterized human FMO2 in terms of genepolymorphism (genotyped by Dr. Hines), protein levels and catalytic activity with humanlung microsomes. An ethnically related polymorphism was observed, in which all Caucasians genotyped to date are homozygous for a truncated, enzymatically inactive enzymewhich may not even be translated. Approximately 15% of humans of African descent are heterozygous forfull- length FMO2, but the level of expression may not be sufficient to significantly effectdrug metabolism in the lung. The results of truncated FMO2 produced from baculovirus expression suggest that the C-terminal of FMO2 might be responsible for enzymestability and/or proper structure necessary to exert fully enzyme activity. We concludethat the human FMO2 possesses unique features compared to all other mammalsexamined to date including other primates. Copyright by Mei-Fei Yueh January 4, 1999 All Rights Reserved IDENTIFICATION AND CHARACTERIZATION OF FLAVIN-CONTAINING MONOOXYGENASE ISOFORM 2 (FMO2) IN RHESUS MONKEY AND EXAMINATION OF A HUMAN FMO2 POLYMORPHISM. by Mei-Fei Yueh A THESIS submitted to Oregon State University in partial fulfillment of the requirement for the degree of Doctor of Philosophy Completed January 4, 1999 Commencement June 1999 Doctor of Philosophy thesis of Mei-Fei Yueh presented on January 4, 1999 APPROVED: Redacted for privacy Major Professor, representing Toxicology Redacted for privacy Head of Depart of Environmental and Molecular Toxicology Redacted for privacy Dean of Gradu School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for privacy Mei-Fei Yueh, Auth ACKNOWLEDGEMENTS I would like to thank the many people who contributed to this project. My parents for supporting me to go abroad and encouraging me in any circumstance. Their encouragement and love were a source of strength to finish this degree.Dr. Williams for his guidance in these studies. He was always very instructive, supportive,patient and encouraging when I was with problems during all these years. Dr. Kruegerfor her technique advise and assistance. It was very helpful having her around as aconsultant for my experiments. Also, people in Dr. Williams Lab:Shelley (Dr. Su), Aram (Dr. Oganesian), Sirinmas, Beth, David (Dr. Carlson), Dustin, Adam and again Sharon(Dr. Krueger). Their techniques support facilitated my whole project, more importantly,their friendship made my life in the Lab much more interesting. My friends: Shu-Huei,John, Lily, Christine, Chu-Liang, Pei-Yu, Pei-Chien, Kung, Kevin, Yea-Huei, Richard, Maryand her family, Sam and his family, my conversant Margaret, and Jeffry, they gave me somuch pleasure in my daily life in Corvallis. This research was supported by NIH grant HL38650. CONTRIBUTION OF AUTHORS Dr. Krueger was involved in the experimental design, technique consultation,and assisted in review of manuscripts. Dr. Hines at Wayne State University (Detroit ,MI). conducted the genotyping for human FMO2 polymorphism in chapter 4. Dr.Williams was involved in the design and analysis of all experiments, especially protein immunoblotting and enzyme assay in chapter 2 and enzyme assays in chapter 4, and in thepreparation of manuscripts TABLE OF CONTENTS Page CHAPTER 1: INTRODUCTION 1 Abstract 2 History 3 Catalytic Mechanism 4 Nomenclature of FMO Gene Family 6 Two Monooxygenase Systems: CYP and FMO 8 Tissue and Cellular Distribution of FMO 10 Role of FMO in Metabolism 11 Regulation of FMO Expression 20 Characterization of FMO Gene Structure 22 Introduction of My Study 24 References 26 CHAPTER 2: PULMONARY FLAVIN-CONTAINING MONOOXYGENASE (FMO) IN RHESUS MACAQUE: EXPRESSION OF FMO2 PROTEIN, mRNA AND ANALYSIS OF THE cDNA Abstract 37 Introduction 38 Materials and Methods 40 Results 44 Discussion 53 Acknowledgements 55 References 56 CHAPTER 3. EXPRESSION OF CLONED FLAVIN-CONTAINING MONOOXYGENASE FROM MONKEY LUNG IN HETEROLOGOUS SYSTEMS Abstract 60 Introduction 61 TABLE OF CONTENTS (Continued) Page Materials and Methods 63 Results 68 Discussion 74 Acknowledgements 76 References 77 CHAPTER 4. IDENTIFICATION OF A HUMAN LUNG FMO2 VARIANT AND CHARACTERIZATION OF TRUNCATED -FMO2 WITH PCR SITE-DIRECTED MUTAGENESIS BY BACULOVIRUS-MEDIATED EXPRESSION Abstract 81 Introduction 82 Materials and Methods 85 Results 91 Discussion 100 References 102 CHAPTER 5. SUMMARY 105 BIBLIOGRAPHY 110 LIST OF FIGURES Figure Page Fig 1.1. Catalytic cycle of FMO Fig 2.1. Nucleotide and derived amino acid sequence from cDNAencoding FMO2 expressed in Rhesus monkey lung 48 Fig 2.2. Primary sequence comparison of FMO2 orthologs 50 Fig 2.3. FMO2 transcripts in Rhesus monkey tissues 51 Fig 2.4. Quantitation of FMO2 mRNA in monkey tissues by RT-PCR 52 Fig 3.1. Immunoblots of expressed proteins 70 Fig 3.2. Immunoblots of purified FMO2 from baculovirus expression 71 Fig 4.1. Human lung FMO2 identified by western blot analysis 95 Fig 4.2. Comparison of FMO2 levels in multiple species 96 Fig 4.3. Catalytic activity with substrate methimazole for expressed FMO2 ... 97 Fig 4.4. Immunoblot of monkey truncated FMO2 98 Fig 4.5. RT-PCR for quantitation of FMO2 mRNA levels 99 LIST OF TABLES Table Page 1.1. Reported Sequences of Flavin-Containing Monooxygenases 7 1.2. Substrates oxidized by FMO 12 2.1. Three pairs of primers used in PCR for FMO2 cDNA cloning 46 2.2. Immunoquantitaion and catalytic activity of FMO2 in primate pulmonary microsome 47 3.1. Protein yield and FMO2 levels in baculovirus-mediated expression 72 3.2. Catalytic activity of expressed FMO2 73 Identification and Characterization of Flavin-Containing Monooxygenase Isoform 2 (FMO2) in Rhesus Monkey and Examination of an Human FMO2 Polymorphism. Chapter 1 INTRODUCTION M.-F. Yueh and D.E. Williams Department of Environmental and Molecular Toxicology Oregon State University, Corvallis OR 2 ABSTRACT Flavin-containing monooxygenase (FMO, EC1.14.13.8) is present in endoplasmic reticulum in a number of tissues of humans and other mammals. FMO comprises a family of xenobiotic-metabolizing enzymes that oxygenate a large number and wide variety of xenobiotics. All FMOs form a kinetically stable 4a-hydroperoxyflavin enzyme intermediate in the presence of NAD(P)H and oxygen, but independent of substrates. Any soft nucleophile accessible to the enzyme-bound peroxy-flavin intermediate will be oxidized. Therefore, the microsomal FMOs catalyze the oxygenation of structurally diverse xenobiotics including amines, sulfides as well as some phosphorous- and selenium- containing compounds. Human, and other mammals, express five different flavin- containing monooxygenases (FMO1, FMO2, FMO3, FMO4, and FMO5) in a species- and tissue- specific manner and each subfamily contains a single gene. The identities among different forms are between 50 and 58% and the identities of all apparent orthologs are 82% or greater. Some of the same substrates are metabolized by both FMO and cytochrome P450, although different metabolites are often produced due to different catalytic mechanisms. In most cases, FMO converts its substrates such as tertiary amines to polar, easily excreted metabolites, although toxic compounds can be generated by FMO metabolism. Human FMOs play an important role in the biotransformation of a variety of chemicals such as the psychoactive drug chlorpromazine, the H2 antagonist cimetidine and the dietary compound trimethylamine. In my study, I characterized the lung specific isoform, FMO2, in monkey which is catalytically active toward many probe substrates and might have a significant role in xenobiotic metabolism in lung. With the information