STRUCTURE AND FUNCTION OF A.NIDULANS PSI FACTOR PRODUCING OXYGENASE A Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades „Doctor rerum naturalium” der Georg-August-Universität Goettingen vorgelegt von Christian Koch aus Mühlhausen Goettingen 2012 Die vorliegende Arbeit wurde in der Zeit von Januar 2010 bis August 2012 am Albrecht-von- Haller-Institut für Pflanzenwissenschaften der Georg-August Universität unter der Anleitung von Prof. Dr. Ivo Feußner angefertigt. Mitglied des Betreuungsausschusses/ Erstreferent: Prof. Dr. Ivo Feußner Abteilung Biochemie der Pflanze, Albrecht-von-Haller-Institut, Georg-August Universität Göttingen Mitglied des Betreuungsausschusses/ Zweitreferentin: Prof. Dr. Marina Bennati Arbeitsgruppe für EPR-Spektroskopie, Max Planck Institut für biophysikalische Chemie, Göttingen Mitglied des Betreuungsausschusses: Prof. Dr. Kai Tittmann Abteilung Bioanalytik, Albrecht-von-Haller-Institut, Georg-August Universität Göttingen Mitglieder der Prüfungskommission: Prof Dr. Ulf Diederichsen Institut für Organische und Biomolekulare Chemie, Georg-August Universität Göttingen Prof. Dr. Ralf Ficner Abteilung für Molekulare Strukturbiologie, Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen Prof. Dr. Heinz Neumann Angewandte Synthetische Biologie, Institut für Mikrobiologie und Genetik, Georg-August Universität Göttingen Tag der mündlichen Prüfung: 01.Oktober 2012 Affidavit Herewith, I declare that the present thesis was written on my own without other sources and aids than quoted. Goettingen, August 2012 Christian Koch STRUCTURE AND FUNCTION OF A.NIDULANS PSI FACTOR PRODUCING OXYGENASE A TABLE OF CONTENTS 1 Introduction ............................................................................................................................ 11 1.1 PpoA: A tale of green molds and red enzymes ..................................................................... 11 1.2 Involvement of oxylipins in host-pathogen interaction ........................................................ 12 1.2.1 Fungal infections and mycotoxin production-A curse for agriculture ........................... 13 1.2.2 The impact of oxylipins in general and Psi-factors in special on fungal pathogenicity .. 15 1.3 A mechanical view on PpoA, an fascinating enzyme to study heme-chemistry ................... 18 1.3.1 Dioxygenase-Domain ..................................................................................................... 19 1.3.2 Cytochrome P450-Domain ............................................................................................. 22 1.4 Interaction of DOX-and P450-enzymes in oxylipin biosynthesis .......................................... 26 2 Objectives and Outline ............................................................................................................ 29 3 Methods .................................................................................................................................. 31 3.1 Protein crystallization ........................................................................................................... 31 3.2 Prediction of protein folds by computer algorithms ............................................................ 34 3.3 Characterizing the oligomeric assembly of a macromolecule by Small-angle X-ray scattering (SAXS) ........................................................................................................................................ 35 3.4 Electron Paramagnetic Resonance and Double Electron Electron Resonance ..................... 37 3.4.1 Double electron-electron-resonance (DEER) ................................................................. 39 3.5 Kinetic Isotope Effects (KIE) .................................................................................................. 41 3.6 Measuring redoxpotentials by direct electro chemistry....................................................... 41 3.7 Identification of oxylipins by LC/MS2 .................................................................................... 42 4 Results ..................................................................................................................................... 45 4.1 Enzyme preparation and quality control .............................................................................. 45 4.2 Attempts to crystallize the enzyme ...................................................................................... 46 4.2.1 Refinement of a previously identified promising condition .......................................... 46 4.2.2 New screening for conditions eventually yielding crystals ............................................ 46 4.2.3 Systematic test of divalent cations ................................................................................ 47 4.2.4 Tryptic digest and/or reductive lysine methylation of PpoA and subsequent crystal screens .................................................................................................................................... 48 4.2.5 GraFix and subsequent crystal screen ............................................................................ 48 4.3 Template-based structure prediction ................................................................................... 51 4.3.1 Predicted structure of PpoA’s DOX-domain and identification of residues putatively involved in dioxygenation ....................................................................................................... 52 4.3.2 Predicted structure of PpoA’s P450-domain and identification of residues putatively involved in hydroperoxy fatty acid rearrangement ................................................................ 55 4.4 Validation of proposed amino acid determinants by biochemical characterization of respective variants ...................................................................................................................... 59 4.4.1 Substrate binding to PpoA’s DOX-Domain may be governed by ionic interaction with Arg336 ..................................................................................................................................... 59 4.4.2 Tyr327: A second tyrosine involved in hydrogen abstraction from the fatty acid substrate’s C8? ......................................................................................................................... 61 4.4.3 Determinants of substrate binding to PpoA’s hydroperoxide-isomerase-domain ........ 63 4.4.4 Asparagine 887 is crucially involved in hydroperoxide rearrangement ........................ 71 4.5 Attempts to convert 5,8-LDS to 7,8-LDS ............................................................................... 74 4.6 Quaternary structure of PpoA .............................................................................................. 76 4.6.1 Measuring the native low-resolution structure of PpoA by SAXS .................................. 76 4.6.2 Measuring tyrosyl-radical distances by DEER ................................................................ 78 4.7 Kinetic isotope effects to probe rate-limiting steps of hydrogen abstraction ...................... 85 4.7.1 Utilized probes ............................................................................................................... 85 4.7.2 Pseudo steady-state kinetics .......................................................................................... 85 4.7.3 End-point measurements ............................................................................................... 89 4.7.4 Is the high KIE for dideuteration at C8 explainable by hydrogen tunneling? ................. 90 4.7.5 Explaining the effect of dideuteration at C5 on oxygen consumption ........................... 91 4.7.6 Stereochemistry of the dioxygenation, catalyzed by PpoA ........................................... 94 4.8 Protein film voltammetry of PpoA ........................................................................................ 95 5 Discussion ................................................................................................................................ 97 5.1 Structure-Function-Relationship ........................................................................................... 97 5.1.1 Active site structure of the DOX-domain and determinants of dioxygenase-activity ... 98 5.1.2 Active site structure of the P450-domain and determinants of hydroperoxy fatty acid isomerase-activity ................................................................................................................. 101 5.1.3 Quaternary structure ................................................................................................... 105 5.2 Kinetic isotope effects to probe the kinetic contribution of hydrogen-transfer steps ....... 107 5.3 Electrochemistry of PpoA ................................................................................................... 111 6 Summary ............................................................................................................................... 113 7 Outlook and future perspectives .......................................................................................... 115 8 Experimental Part .................................................................................................................. 117 8.1 Wet lab methods................................................................................................................