A Dissertation Entitled Porcine Leukocyte 12-Lipoxygenase By Shu Xu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry Dr. Max O. Funk, Jr., Committee Chair Dr. Douglas W. Leaman, Committee Member Dr. Timothy C. Mueser, Committee Member Dr. Steven J. Sucheck, Committee Member Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo May 2012 Copyright © 2012, Shu Xu This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Porcine Leukocyte 12-Lipoxygenase by Shu Xu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry The University of Toledo May 2012 Lipoxygenases are a class of non-heme, non-sulfur iron dioxygenases in polyunsaturated fatty acid metabolism involved in the modulation of basic physiologic processes. The work described within this dissertation covers biochemical characterization of porcine leukocyte 12-lipoxygenase, and presents the crystal structure of its catalytic domain. For the first time, the iron content of the full length protein was obtained as high as 0.94 atom per molecule. The loss of this iron atom from the protein was evident, as the pH declined from 5 to 4 without losing the native protein folding, by electrospray ionization mass spectroscopy measurements. The full length protein failed to yield crystals in the screening conditions using numerous site-directed mutagenesis experiments and multiple crystallization techniques. As part of this study, the catalytic domain of porcine leukocyte 12-lipoxygenase was successfully expressed in E. coli cells. The crystal structure of porcine 12-lipoxygenase iii catalytic domain was determined to 1.89 Å as a complex with its specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) (PDB id: 3RDE). This represents the first structural description of the 12-lipoxygenase catalytic domain. The complex revealed a new one-open-end U-shaped channel for the natural substrate, arachidonic acid, which is remarkably different from the inhibitor (RS75091) binding pocket of rabbit 15-lipoxygenase. Crystallization of the 12-lipoxygenase catalytic domain with arachidonic acid was also performed. Only fragments of electron density in the active site were present in the structure of the substrate-enzyme complex, illustrating that the substrate is not conformationally fixed in the crystal and may be converted into its products and derivatives. In addition, an unbound form of the catalytic domain was observed in the crystal structure of a recombinant manganese reconstituted lipoxygenase catalytic domain. Almost no structural differences were observed between the bound and unbound forms suggesting that lipoxygenase catalytic domain has little structural fluctuation or conformational alteration upon the binding of the inhibitor or substrate. This research resulted in detailed biochemical insights for recombinant porcine leukocyte 12-lipoxygenase and its catalytic domain. The crystal structures of 12-lipoxygenase catalytic domain revealed key structural features of this enzyme, and also provided a basis for understanding enzymatic catalysis. iv To my wife, Hongyan Ma, for your endless love. To my parents and parents-in-law, for your support and understanding the importance of higher learning. To my lovely son, Shaun Xu, for the happiness you bring into my life. Acknowledgements First, I would like to address special thanks to my advisor, Dr. Max Funk, for all his support, advice, endless patience and continuous encouragement during these years. I would also like to thank Dr. Douglas Leaman, Dr. Timothy Mueser, and Dr. Steven Sucheck for their service in my advisory committee. I acknowledge Dr. Lawrence Marnett at Vanderbilt University for providing the cDNA of 12-lipoxygenase and the inhibitor used in this dissertation. Thanks go to Dr. Timothy Mueser, Dr. Alexander Pavlovsky and Dr. Leif Hanson for their assistances with X-ray crystallographic techniques; to Dr. Pannee Burckel for the measurements of atomic absorption; to Dr. Wendell Griffith and Dr. Dragan Isailovic for electrospay ionization measurements. I would like to thank the staff members at LS-CAT at the Advanced Photon Source of Argonne National Laboratory for their help with X-ray diffraction data collection. I would like to express thanks to former and current members of Dr. Funk’s group: Dr. Allan Sharp, Johanna Rapp, Ilka Decker, Maureen Gibbs, Marie Miniear, Kathryn Guinta, and Waqar Arif. Thanks to electronics specialists Thomas Kina and Youming Cao. I also extend appreciation to our neighbors at the University of Toledo, Dr. Viola’s, Dr. Mueser’s, and Dr. Leaman’s groups and all others for letting me using their equipment, as well as providing a wonderful work environment. vi Table of Contents Abstract…………………………………………………………………………………...iii Acknowledgements………………………………………………………………………vi Table of Contents………………………………………………………………………vii List of Tables……………………………………………………………………………xi List of Figures…………………………………………………………………...………xii List of Abbreviations……………………………………………………………………xv 1 Lipoxygenase: Structure, Biochemistry, and Biological Functions…………………1 1.1 Introduction…………………………………………………………………1 1.2 Structures of lipoxygenases…………………………………………………4 1.3 Lipoxygenase catalytic reactions……………………………………………7 1.3.1 Dioxygenase activity of lipoxygenases…………………………………7 1.3.2 Other enzymatic activities of lipoxygenases…………………………11 1.4 Metabolites from lipoxygenase reactions………………………………….12 1.4.1 Leukotriene pathway…………………………………………………15 1.4.2 Lipoxin pathway………………………………………………………17 1.4.3 Hepoxilin pathway…………………………….………………………18 1.4.4 Peroxide reduction pathway…………………………………………20 vii 1.5 Biological functions of lipoxygenase metabolites…………………………21 1.5.1 Roles of leukotrienes through 5-LOX pathway……………………….21 1.5.2 Roles of metabolites via 12/15-LOX pathway………………………22 1.5.2.1 Biological functions of lipoxins…………………………………23 1.5.2.2 Biological function of hepoxilins…………………………………23 1.5.3 Biological functions of HETEs and derivatives………………………24 1.6 Perspectives………………...………………………………………………25 2 Experimental Methods………………..……………………………………………..27 2.1 Mutagenesis of plasmids of porcine leukocyte 12-lipoxygenase………….27 2.2 Construction of plasmids of 12-lipoxygenase catalytic domain…………...28 2.3 Expression of 12-lipoxygenase and its mutants……………………………29 2.4 Expression of 12-lipoxygenase catalytic domain…………………….29 2.5 Expression of recombinant manganese 12-lipoxygenase catalytic domain……………………………………………………………………29 2.6 Purification of 12-lipoxygenase and 12-lipoxygenase catalytic domain…..30 2.7 Lipoxygenase enzymatic assay……………………………………………31 2.8 Protein concentration determination……………………………………….31 2.9 Sodium dodecyl sulfate polyacrylamide gel electrophoresis………………32 2.10 Atomic absorption measurements…………………………………………32 2.11 Electron ionization mass spectroscometry measurements…………………33 2.12 Ellman’s tritration…………………………………………………………33 viii 2.13 Dynamic light scattering measurements…………………………………34 2.14 Optimization of crystallization conditions…………………………………35 2.15 Crystallization of HLCDS-OPP and Mn-HLCDS-OPP……………………35 2.16 Crystallization of HLCDS-AA complex…………………………………...36 2.17 Diffraction data collection and structure determination…………………36 3 Biochemical Characterization of Porcine Leukocyte 12-Lipoxygenase………….…38 3.1 Introduction………………………………………………………………38 3.2 Results……………………………………………………………………39 3.2.1 Expression and purification of 12-lipoxygenase………………………39 3.2.2 Iron content of 12-lipoxygenase………………………………………41 3.2.3 ESI-MS measurements of 12-lipoxygenase…………………………...42 3.2.4 Crystallization experiments of 12-lipoxygenase………………………43 3.2.5 Protein engineering mutation to enhance crystallizability of PLL……45 3.2.6 Crystal screening results of PLL mutants……………………………48 3.3 Discussion…………………………………………………………………49 4 Crystal Structures of 12-Lipoxygenase Catalytic Domain………..……………52 4.1 Introduction………………………………………………………………52 4.2 Results……………………………………………………………………53 4.2.1 Expression of 12-lipoxygenae catalytic domain………………………53 4.2.2 Crystallization of 12-lipoxygenae catalytic domain…………………..55 4.2.3 Data collection and structure determination of HLCDS-OPP ix complex………………………………………………………………56 4.2.4 Specific inhibitor in the structure of 12-lipoxygenae catalytic domain…………………………………………………………………60 4.2.5 Expression of recombinant manganese 12-lipoxygenase catalytic domain…………………………………………………………………62 4.2.6 Crystallization of Mn-HLCDS-OPP and HLCDS–AA complexes……62 4.2.7 Data collection and structure determination of Mn-HLCDS-OPP and HLCDS-AA complexes……………………………………………….65 4.2.8 An inhibitor unbound form of 12-lipoxygenase catalytic domain from the crystal structure of Mn-HLCDS-OPP complex…………………65 4.2.9 The position of AA in HLCDS-AA complex………………………….67 4.3 Discussion…………………………………………………………………68 4.3.1 Crystallization conditions for 12-lipoxygenase catalytic domain……68 4.3.2 Variation of helix α2 in mammalian lipoxygenases…………………...69 4.3.3 The iron sites of mammalian lipoxygenases…………………………71 4.3.4 The inhibitor binding pockets of mammalian lipoxygenases…………72 4.3.5 Implication for inhibition and enzymatic catalysis……………………73 4.3.6 Substrate/intermediate binding site in 12-lipoxygenase………………76 5 Conclusions………………………………………………………………………….79 References………………………………………………………………………………..83 x List of Tables 1.1 Iron environment of lipoxygenases………………………………………………….6 2.1