Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage oxygenases DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Carlo C. dela Seña The Ohio State Biochemistry Program The Ohio State University 2014 Dissertation Committee: Earl H. Harrison, Ph.D., Adviser Robert W. Curley, Jr., Ph.D. Ross E. Dalbey, Ph.D. Steven J. Schwartz, Ph.D Copyright by Carlo C. dela Seña 2014 i Abstract Carotenoids are yellow, orange and red pigments found in fruits and vegetables. Some carotenoids can act as dietary precursors of vitamin A. Humans and other animals generate retinal (vitamin A aldehyde) from provitamin A carotenoids by oxidative cleavage of the central 15-15′ double bond by the enzyme β-carotene 15-15′-oxygenase (BCO1). Another carotenoid oxygenase, β-carotene 9′-10′-oxygenase (BCO2), catalyzes the oxidative cleavage of the 9′-10′ double bond of various carotenoids to yield apo-10′- carotenals and ionones. In this dissertation, we elucidate the substrate specificity of these two enzymes. Recombinant His-tagged human BCO1 was expressed in Escherichia coli strain BL21- Gold (DE3) and purified by cobalt ion affinity chromatography. The enzyme was incubated with various dietary carotenoids and β-apocarotenals, and the reaction products were analyzed by reverse-phase high-performance liquid chromatography (HPLC). We found that BCO1 catalyzes the oxidative cleavage of only provitamin A dietary carotenoids and β-apocarotenals specifically at the 15-15′ double bond to yield retinal. A notable exception is lycopene, which is cleaved by BCO1 to yield two molecules of acycloretinal. Previous studies have found lycopene to be unreactive with BCO1. Our results warrant a fresh look at acycloretinal and its alcohol and acid forms as possible ii metabolites of lycopene. We also found that BCO1 does not react with 9-cis-β-carotene. It has been previously suggested the 9-cis-retinoic acid, a ligand of retinoid X receptors (RXR’s), is generated by BCO1 cleavage of 9-cis-β-carotene to 9-cis-retinal and subsequently oxidized to the acid. However, our results strongly argue against this. Similarly, the substrate specificity of purified recombinant chicken BCO2 was also tested by incubating purified enzyme with the test substrates and analysis of the products by HPLC. Unlike BCO1, BCO2 reacts with full length provitamin A carotenoids as well as non-provitamin A carotenoids that contain 3-hydroxy ionone rings. However, it does not react with lycopene and β-apocarotenals. Carotenoid cleavage oxygenases (CCO’s) oxidatively cleave carotenoids to yield aldehydes and/or ketones. Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase based on a study that used conditions that favored oxygen exchange with water. We elucidated the reaction mechanism of BCO1 using the same principles of oxygen labeling experiments, but minimized the reaction and processing times to minimize oxygen exchange between retinal and water. We incubated purified 16 18 recombinant human BCO1 and β-carotene in an O2-H2 O medium for 15 minutes at 37°C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 18O-retinal makes up only 3-10% of the total retinal. Incubation of 16O-retinal under the same conditions yields 5- 18 18 16 13% O-retinal. We also incubated BCO1 and β-carotene in an O2-H2 O medium. Under these conditions, 18O-retinal makes up 79-85% of the total retinal product. iii Similarly, incubation of 91% 18O-retinal under the same conditions yields 67-84% 18O- retinal. Our results show that BCO1 incorporates only oxygen from O2 into retinal, and BCO1 is therefore a dioxygenase. iv Acknowledgments I would like to express my deepest gratitude to my adviser, Dr. Earl H. Harrison. Your guidance, support and encouragement made me grow as a scientist and as a person. Thank you for believing in me and for always finding something good in my work, even during those times when I am disappointed in myself. I am eternally grateful for everything I learned from you. Thanks to my committee members, Dr. Robert W. Curley Jr., Dr. Steven J. Schwartz and Dr. Ross E. Dalbey, for their thoughtful comments that guided this work. I would also like to thank Dr. Curley and Dr. Schwartz for allowing me to work in their laboratories, and sharing their wisdom and resources. Thanks also to the members of the Schwartz and Curley labs for attending to all my needs and inquiries. I am grateful to Dr. Sureshbabu Narayanasamy for the excellent work on the synthesis of organic compounds for my projects, and to Dr. Kenneth M. Riedl for his outstanding expertise in mass spectrometry. Thanks to the past and present members of the Harrison laboratory who worked with me- Dr. Matthew K. Fleshman, Dr. Abdulkerim Eroglu, Dr. Jian Sun, Vanessa Reed, Shiva Raghuvanshi, Yan Yuan, and Sara Thomas. Thanks for helping me in the lab and for all the wonderful meals and conversations. v Special thanks to my first lab mates-Dr. Jason D. Fowler, Dr. Shanen Sherrer and Dr. Cuiling Xu-for the training in the techniques that had been very useful to me in my research. I am forever thankful to my friends in Columbus for being there for me through the best and worst of times. Thanks to my family for inspiring me to make the best of what I have. Thanks to The Ohio State University and the United States of America for giving me this wonderful opportunity. vi Vita 1999-2003 ....................... B.S. Chemistry, Mindanao State University-Main Campus, Philippines 2003-2005 ...................... Instructor, University of the Philippines at Los Baños, Philippines 2005-2006 ...................... Chemist, Central Analytical Laboratory, San Miguel Corporation-Beer Division, Philippines 2006-2008 ...................... M.S. Chemistry Program (completed 24 units), De La Salle University-Manila, Philippines Lecturer (part-time), De La Salle University-Manila, University of the Philippines-Manila, Manila, Philippines 2008-2009, 2010-2014 .. Graduate Research Associate, The Ohio State University 2009-2010 ....................... Graduate Teaching Associate, The Ohio State University 2013-2014 ....................... Food Innovation Center Doctoral Research Grant 2013 ................................. Poster Award Winner, Carotenoids Research Interaction Group (CARIG)/Vitamin A Research Interaction Group (VARIG) Poster Competition Publications Eroglu, A., Hruszkewycz, D. P., dela Seña, C., Narayanasamy, S., Riedl, K. M., Kopec, R. E., Schwartz, S. J., Curley, R. W., Jr., and Harrison, E. H. (2012) Naturally-occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors. J. Biol. Chem. 287, 15886-15895 Harrison E.H., dela Seña, C., Eroglu A., Fleshman M.K. (2012) The formation, occurrence, and function of β-apocarotenoids: β-carotene metabolites that may modulate nuclear receptor signaling. Am. J. Clin. Nutr. 96:1189S-92S dela Seña, C., Narayanasamy, S., Riedl, K. M., Curley, R. W. Jr. Schwartz, S. J., and Harrison, E. H. (2013). Substrate specificity of purified recombinant human β-carotene 15,15'-oxygenase (BCO1). J. Biol. Chem. 288, 37094-37103 vii dela Seña, C. Riedl, K. M., Narayanasamy, S., Curley, R. W. Jr. Schwartz, S. J., and Harrison, E. H. (2013). The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase. J. Biol. Chem. Published online March 25, 2014 Field of Study Major Field: Biochemistry viii Table of Contents Abstract .....................................................................................................................................ii Acknowledgments .................................................................................................................... v Vita ..........................................................................................................................................vii Table of Contents .................................................................................................................... ix List of Abbreviations .............................................................................................................xii List of Tables .........................................................................................................................xiv List of Figures......................................................................................................................... xv Chapter 1: Literature Review ................................................................................................. 1 1.1 Introduction .................................................................................................................... 2 1.2 Strucure and metabolism of dietary carotenoids .......................................................... 2 1.3 Carotenoid cleavage oxygenases................................................................................... 5 1.4 β-Carotene 15-15′-oxygenase ...................................................................................... 10 1.5 β-Carotene 9′-10′-oxygenase ....................................................................................... 13 1.6 Tables ............................................................................................................................ 16 1.7