Recombinant expression and characterization of a prokaryotic lipoxygenase from Pseudomonas aeruginosa. Investigations into the biological role of this enzyme Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by SWATHI BANTHIYA from Bangalore, India 2017 This work was carried out between 01.01.2014 and 16.02.2017 at the Institute for Biochemistry, Universitätsmedizin Berlin Charitè, under the supervision of Prof. Dr. sc. Med. Hartmut Kühn. 1st Reviewer: Prof. Rudolf Tauber 2nd Reviewer: Prof. Hartmut Kühn Date of defence: 22.06.2017 Acknowledgement I ACKNOWLEDGEMENT None of us got to where we are alone. Whether the assistance we received was obvious or subtle, acknowledging someone's help is a big part of understanding the importance of saying thank you. - Harvey Mackay It has been a wholly gratifying experience, but the vast majority of it were the joy I received by working with Prof. Hartmut Kühn. He has been a great mentor. I am honored to have worked under him. He is an amazing teacher who made the journey of my doctoral studies enjoyable and at the same time extremely productive. I have learnt a lot from him that will stay with me for the rest of my life. He was always there when we needed his time and made sure we always excelled in anything we attempted to venture into. Prof. Rudolf Tauber actively took interest in the progress of my project throughout. His valuable feedback and suggestions always motivated me to work better. I am grateful for his assistance and being a catalyst for achieving my goal. I am thankful to Dr. Dagmar Heydeck, Dr. Astrid Borchert and Dr. Christoph Ufer for their excellent scientific guidance throughout the tenure of my doctoral study and for constantly encouraging me in the most optimistic manner towards achieving my goals. A special mention goes to Mrs. Sabine Stehling, for her excellent technical guidance and for always patiently guiding me with new techniques. A memorable note to Maria, Marlena, Eugenia, Felix, Sajad, Sophia, and Ilya for being a part of and sharing my learning experiences. In addition, I extend my thanks to Dr. Patrick Scheerer, Jacqueline Kalms, Etienne Galemou Yoga, Dr. Valerie O’Donnell, Dr. Maceler Aldrovandi, Prof. Mats Hamberg, Prof. Dr. Hermann-Georg Holzhütter and Dr. Igor Ivanov for their successful collaboration throughout the duration of my study. To my parents, Dr. Ummed Singh Banthiya and Mrs. Surajkala Banthiya, I am forever indebted. My biggest supporters, Sandeep and Shruthi, a huge applause. To my husband, Aakash, my north star, I don’t have enough words to express my gratitude to him. Lots of love to my nephews for being the best stress-busters ever. There are a lot more people who have helped me to reach where I am today and I would like to include all of them in my grace for being a part of my life and fueling my ambition. Table of contents II TABLE OF CONTENTS ACKNOWLEDGEMENT ..................................................................................... I LIST OF ABBREVIATIONS ............................................................................. VII 1. INTRODUCTION ............................................................................................ 1 1.1. Lipoxygenases ............................................................................................ 1 1.1.1. History of lipoxygenases ........................................................................... 1 1.1.2. Distribution of lipoxygenases in terrestrial life ........................................... 3 1.1.3. Lipoxygenase reaction.............................................................................. 6 1.1.4. Nomenclature of lipoxygenases ............................................................... 7 1.2. Biological role of LOX-isoforms ................................................................... 8 1.2.1. In mammals .............................................................................................. 8 1.2.2. In plants .................................................................................................. 11 1.2.3. In prokaryotes ......................................................................................... 12 1.3. Properties of lipoxygenases ...................................................................... 14 1.3.1. Protein-chemical properties of lipoxygenases ........................................ 14 1.3.1.1. Structural properties of lipoxygenases ................................................. 14 1.3.1.2. Mutant variants of lipoxygenases ........................................................ 16 1.3.2. Enzymatic properties of lipoxygenases .................................................. 20 1.3.2.1. Reaction conditions favored by different LOX-isoforms ....................... 20 1.3.2.2. Substrate specificity of lipoxygenases ................................................. 22 1.3.2.3. Reaction stereochemistry of lipoxygenases ........................................ 25 1.4. Aims of this project .................................................................................... 27 2. MATERIALS AND METHODS ..................................................................... 28 2.1. Materials .................................................................................................... 28 2.1.1. Chemicals ............................................................................................... 28 2.1.2. Fatty acid substrates .............................................................................. 29 2.1.3. Phospholipid substrates ......................................................................... 29 2.1.4. HPLC-standards ..................................................................................... 30 2.1.5. Lipoxin and Leukotriene synthesis ......................................................... 30 2.1.6. Cells and media ...................................................................................... 30 Table of contents III 2.1.7. Rabbit ALOX15 ...................................................................................... 31 2.1.8. Buffers .................................................................................................... 32 2.1.9. Kits ......................................................................................................... 32 2.2. Methods ..................................................................................................... 32 2.2.1. Site directed mutagenesis ...................................................................... 32 2.2.2. Protein expression .................................................................................. 34 2.2.3. Quantification of protein concentration and of the degree of purity ........ 36 2.2.4. Interaction of PA-LOX with different classes of substrates ..................... 37 2.2.4.1. Fatty acid oxygenase activity ............................................................... 37 2.2.4.2. Phospholipid oxygenase activity .......................................................... 38 2.2.4.3. Oxygenation of membrane bound phospholipids ................................ 39 2.2.4.4. Oxygenation of intact erythrocyte membranes .................................... 39 2.2.4.5. Leukotriene and lipoxin synthesis ........................................................ 40 2.2.5. Activity assay .......................................................................................... 41 2.2.5.1. Spectrophotometric analysis ............................................................... 41 2.2.5.1.1. PA-LOX with different PUFAs ........................................................... 41 2.2.5.1.2. Temperature profile .......................................................................... 41 2.2.5.1.3. Variable oxygen concentrations ....................................................... 41 2.2.5.1.4. Experiments with stereospecifically labeled substrates .................... 43 2.2.5.2. Oxygraphic activity assay .................................................................... 43 2.2.5.3. HPLC quantification of reaction products ............................................ 44 2.2.5.3.1. Reversed phase - HPLC (RP-HPLC) ................................................ 44 2.2.5.3.2. Straight phase - HPLC (SP-HPLC) ................................................... 45 2.2.5.3.3. Chiral phase - HPLC (CP-HPLC)...................................................... 46 2.2.6. Storage stability of purified enzyme ........................................................ 46 2.2.7. Determination of iron content ................................................................. 47 2.2.8. GC/MS and LC/MS/MS analysis ............................................................ 47 2.2.8.1. Analysis of fatty acid oxygenation products of PA-LOX ....................... 47 2.2.8.2. Analysis of products with stereospecifically labeled substrates ........... 47 2.2.8.3. Oxidized lipids of PA-LOX treated RBCs ............................................. 48 2.2.9. Protein crystallization.............................................................................. 49 Table of contents IV 2.2.9.1. Wildtype- PA-LOX ............................................................................... 49 2.2.9.2. Ala420Gly-PA-LOX.............................................................................. 51 3. RESULTS ....................................................................................................