UPDATE ON THE DISTRIBUTION AND EVOLUTION OF THE ALDEHYDE DEHYDROGENASE SUPERFAMILY IN VERTEBRATES AND BIOCHEMICAL AND POLYMORPHIC CHARACTERIZATION OF HUMAN ALDH1B1 by BRIAN CHRISTOPHER JACKSON B.S., University of California, Riverside 2004 M.S., University of Texas at Tyler, 2007 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Toxicology Program 2015 This thesis for the Doctor of Philosophy degree by Brian Christopher Jackson has been approved for the Toxicology Program by Dennis Petersen, Chair Vasilis Vasiliou, Advisor David Bain David Orlicky David Thompson Date 5/07/2015 ii Jackson, Brian Christopher (Ph.D., Toxicology) Update on the Distribution and Evolution of the Aldehyde Dehydrogenase Superfamily in Vertebrates and Biochemical and Polymorphic Characterization of Human ALDH1B1 Thesis directed by Professor Vasilis Vasiliou ABSTRACT The aldehyde dehydrogenase (ALDH) superfamily is a group of enzymes that catalyze the NAD(P)+-dependent oxidation of a wide variety of endogenous and exogenous aldehydes to their corresponding carboxylic acids. This family is present in all taxonomic lineages studied, including archaea, bacteria, and eukaryotes. As a torrent of new genomic data has become available over the past decade, there is now a need to organize and examine the evolution of this critical superfamily. To create a reference of the distribution and number of ALDHs in vertebrates, 11 representative species with completed genomes were examined and the full number of ALDHs was manually studied (Chapter II). One recently investigated gene, ALDH1B1 appeared to have a limited distribution and high similarity to ALDH2. This gene has received increased attention recently as a mediator of alcohol metabolism, growth and development, and as a biomarker and possibly key mediator of colon cancer. The complete known distribution of ALDH1B1 was investigated, as well as its evolutionary origins as a retrotransposition of ALDH2. In addition, it was shown that although ALDH1B1 has a unique pattern of expression and substrate specificity from ALDH2, they retain enough similarity that heterotetramerization (and possibly cross-regulation) may be feasible (Chapter III). From iii determining the distribution of ALDH1B1 and ALDH16A1 across phylogenies, in both cases frogs appeared to have unusual patterns of ALDH distributions. Since there was no frog representative in previous work, the full number of frog ALDHs was determined and full gene trees were created to examine the phylogenetic distribution of frog ALDHs. This also allowed deeper examination of the distribution and evolution of ALDHs. From this analysis it was determined rather than frogs being unusual, ALDH1B1 likely arose in the early vertebrate lineage and was subsequently lost in species other than frogs and mammals, and that a unique non-catalytic version of ALDH16A1 likely arose in fish, and was transferred to an early amniote ancestor (Chapter IV). This and other examples of evolution of ‘dead-enzymes’ within enzyme families led to the search for additional examples of non-catalytic ALDHs. 182 examples were found across all three kingdoms, which were divided into 19 groups based on protein sequence, with a large number of newly discovered records coming from bacteria and fungi (Chapter IV). Finally, the substrate specificity and effect of human polymorphisms of ALDH1B1 were investigated in depth, and it was found that ALDH1B1 likely plays a role in growth and development via retinaldehyde metabolism, and that this function may be disrupted by mutations prevalent in human populations, especially via the ALDH1B1*2 (A86V) mutation (Chapter V). This work together enhances our understanding of the distribution and evolutionary origins of the ALDH superfamily as a whole, and increases the understanding of the mechanisms of action of ALDH1B1 in particular. The form and content of this abstract are approved. I recommend its publication. Approved: Vasilis Vasiliou iv ACKNOWLEDGEMENTS I would like to thank the current and former members of the Vasiliou lab for their help and support over the years. First I appreciate the efforts of Elizabeth Donald and Bettina Miller for keeping the lab organized, stocked and compliant. In addition, many lab members helped, advised, or worked with me during the past years including Ying Chen, Chad Brocker, Guarav Mehta, Surendra Singh, Vindhya Koppaka, Akiko Matsumoto, Monica Sandoval, and Claire Heit. I would also like to thank the labs that I did research / training rotations in including the lab of Richard Radcliffe and David Ross, especially the training from Chao Yan, David Siegel, and J. ‘Gigi’ Kepa. In addition, I would like to acknowledge the guidance and assistance from my advisor, Vasilis Vasiliou and my committee, Dennis Petersen, David Thompson, David Orlicky, and David Bain. Many thanks go out to my first mentor Blake Bextine and the people I worked with both at UC Riverside and UT Tyler, for all of the work and guidance that they gave to get me to where I am today. I appreciate the support and love of my family including my Mom and Dad, brothers and sister, in-laws, nephews, nieces, and all of the extended group that I consider home. I appreciate the patience and support of my wife Natalie Vitovsky, and my ever-constant companions Rupert and Stella. I would also like to acknowledge NRSA fellowship support from the NIAAA (F31 AA020728). v TABLE OF CONTENTS CHAPTER I. INTRODUCTION………………………………………………………………...1 The ALDH Superfamily…………………………………………………..1 Distribution of ALDH Genes in Vertebrates…………………………….10 Frog ALDHs and Phylogenies of Vertebrate ALDHs…………………...12 Evolution and Structural Similarities between ALDH1B1 and ALDH2...14 ALDH ‘Dead Enzymes’………………………………………………….17 Substrate Specificity and Human Mutations of ALDH1B1……………..26 II. UPDATE ON THE ALDEHYDE DEHYDROGENASE GENE (ALDH) SUPERFAMILY IN VERTEBRATES………………………………………….31 Summary…………………………………………………………………31 Introduction………………………………………………………………32 Methods………………………………………………………………..…35 Results…………………………………………………………………....37 Discussion………………………………………………………………..58 III. UPDATE ON THE ALDEHYDE DEHYDROGENASE GENE (ALDH) SUPERFAMILY IN FROG (XENOPUS TROPICALIS) – AN EXAMPLE OF POSSIBLE HORIZONTAL GENE TRANSFER……………………………….64 Summary………………………………………………………………....64 Introduction……………………………………………………………....65 Methods…………………………………………………………………..67 Results…………………………………………………………………....68 Discussion………………………………………………………………..77 IV. COMPARATIVE GENOMICS, MOLECULAR EVOLUTION AND COMPUTATIONAL MODELING OF ALDH1B1 AND ALDH2……………..80 Summary…………………………………………………………………80 vi Introduction……………………………………………………………....81 Methods………………………………………………………………..…84 Results……………………………………………………………………86 Discussion………………………………………………………………104 V. ROLE OF DEAD ENZYMES OF THE ALDEHYDE DEHYDROGENASE FAMILY IN DRUG METABOLISM AND TOXICOLOGY…………………106 Summary……………………………………………………………..…106 Introduction…………………………………………………………..…107 Discovering new ALDH dead-enzymes………………………………..109 Discussion………………………………………………………………111 VI. HUMAN ALDH1B1 POLYMORPHISMS MAY AFFECT THE METABOLISM OF ACETALDEHYDE AND ALL-TRANS RETINALDEHYDE – IN VITRO STUDIES AND COMPUTATIONAL ……………………………………...…123 Summary………………………………………………………………..123 Introduction……………………………………………………………..124 Methods…………………………………………………………………128 Results…………………………………………………………………..136 Discussion………………………………………………………………152 VII. SUMMARY AND FUTURE DIRECTIONS…………………………………..161 REFERENCES………………………………………………………………………....165 vii LIST OF TABLES TABLE 2.1 Total number of aldehyde dehydrogenase (ALDH) NCBI gene records identified within each species’ genome…………………………………………………….39 2.2 ALDH genes and duplicated genes across species with respective chromosome (Chr) locations…………………………………………………………………...43 2.3 List of the Entrez Gene ID (GI), chromosome location, presence of introns, gene type and recommended gene name of all ALDH genes in this study that show evidence of gene duplication, compared with that in the human genome……….45 2.4 Tabulation of all ALDH genes in this study that show evidence of gene duplication, compared with that in the human genome………………………….47 2.5 Known copy number variations in humans……………………………………...55 3.1 Frog ALDH genes………………………………………………………………..70 3.2 Exons present in ALDH16A1 by species………………………………………..77 4.1 ALDH1B1 and ALDH1A genes and enzymes in selected vertebrate species…...88 4.2 Aldehyde dehydrogenase (ALDH2) genes and enzymes in selected vertebrate species……………………………………………………………………………89 4.3 Comparative docking interaction energies and protein stabilities for ALDH2 and ALDH1B1 subunits…………………………………………………………….100 4.4 Specific interactions made by ALDH homo- and heterotetramers……………..103 5.1 Non-enzymatic functions of ALDHs…………………………………………...109 5.2 Summary of groups of ALDH dead enzyme records…………………………...114 5.3 Full list of ALDH dead enzyme records………………………………………..115 5.4 Summary of mutations of key residues for ALDH dead enzyme groups………121 6.1 Computational modeling of interactions between ALDH isozymes and substrates………………………………………………………………………..138 6.2 Kinetic values for the metabolism of select substrates by ALDH isozymes…...141 6.3 Polymorphisms of human ALDH1B1, and variant frequency by race…………143 6.4 Summary of docking poses for NAD+ binding to ALDH isozymes……………147 viii 6.5 Root mean square (RMSD) distances between ALDH1B1 variants and wild- type……………………………………………………………………..………150 6.6 Homology modeling metrics for ALDH1B1 and variants……………………...150