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Structure and Function of Enzymes Involved in Tetrapyrrole Biosynthesis

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Structure and Function of Enzymes Involved in Tetrapyrrole Biosynthesis Structure and Function of Enzymes Involved in Tetrapyrrole Biosynthesis Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Ilka Ursula Heinemann aus Osnabrück 1. Referent: Prof. Dr. Dieter Jahn 2. Referent: Prof. Dr. Michael Steinert eingereicht am: 20.08.2007 mündliche Prüfung (Disputation) am: 21.09.2007 Druckjahr 2007 VORVERÖFFENTLICHUNGEN DER DISSERTATION Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für Lebenswissenschaften, vertreten durch den Mentor der Arbeit in folgenden Beiträgen vorab veröffentlicht: PUBLIKATIONEN Buchenau, B., Kahnt, J., Heinemann, I.U., Jahn, D., Thauer, R.K. Heme biosynthesis in Methanosarcina barkeri via a pathway involving two methylation reactions. J. Bacteriol. 2006 Dec;188(24):8666-8. Heinemann, I.U., Diekmann, N., Masoumi, A., Koch, M., Messerschmidt, A., Jahn, M., Jahn, D. Functional definition of the tobacco protoporphyrinogen IX oxidase substrate- binding site. Biochem. J. 2007 Mar 15;402(3):575-80. EINGEREICHTE PUBLIKATIONEN Tan, F.C., Cheng, Q., Saha, K., Heinemann, I.U., Jahn, M., Jahn, D. and Smith, A.G. Identification of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase by functional complementation of a yeast mutant. Under revision in Biochemical Journal. Masoumi, A., Heinemann, I.U., Rhode, M., Jahn, M., Jahn, D. Metabolic channeling of photoreactive protoporphyrinogen IX in Thermosynechococcus elongatus during the ultimate steps in porphyrin biosynthesis. Under revision in Journal of Bacteriology. Heinemann, I.U., Schulz, C., Jaffe, E., Schubert, W.D., Wachi, M., Jahn, M., Jahn, D. Structure of the Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin. Under review. TAGUNGSBEITRÄGE Heinemann, I.U., Schulz, C., Jaffe, E., Schubert, W.D., Wachi, M., Jahn, M., and Jahn, D. The molecular target of the antibiotic alaremycin. Implications from ist crystal structure with porphobilinogen synthase and kinetic parameters. (Poster) Gordon Research Conference, Enzymes, Coenzymes and Metabolic Pathways, Biddeford, USA. (2007) Heinemann, I.U., Schulz, C., Jaffe, E., Schubert, W.D., Wachi, M., Heinz, D., Jahn, M., and Jahn, D. Crystal structure of Pseudomonas aeruginosa porphobilinogen synthase (PBGS) in complex with the novel antibiotic alaremycin. (Poster) VAAM-Jahrestagung, Osnabrück, Germany. (2007) Heinemann, I.U., Diekmann, N., Masoumi, A., Koch, M., Jahn, M. and Jahn, D. Functional definition of the active site of protoporphyrinogen IX oxidase involved in tetrapyrrole biosynthesis. (Poster) VAAM-Jahrestagung, Jena, Germany. (2006) Heinemann, I.U., Jahn, M., Moser, J., Schubert, W.D., Heinz, D., Warren, M., Thauer, R.K. and Jahn, D. Towards the elucidation of the late pathway to heme. (Poster) Gordon Research Conference, Archaea: Ecology, Metabolism & Molecular Biology, Oxford, Great Britain. (2006) Heinemann, I.U., Jahn, M. and Jahn, D. Structure-based mutagenesis of tobacco protoporphyrinogen IX oxidase. Porphyrins and Porphyrias. (Poster) Cape town, South Africa. (2005) Meiner Familie „Nicht Kunst und Wissenschaft allein, Geduld muss bei dem Werke sein.“ Johann Wolfgang von Goethe Faust, Der Tragödie erster Teil, Hexenküche Table of Contents TABLE OF CONTENTS ABBREVIATIONS ..................................................................................................................III 1 INTRODUCTION ............................................................................................................ 6 1.1 Tetrapyrroles.........................................................................................................................................6 1.1.1 Structure and Function of Naturally Occurring Tetrapyrroles.......................................................6 1.1.2 Biosynthesis of Tetrapyrroles........................................................................................................8 1.1.3 Heme Biosynthesis ........................................................................................................................9 1.1.4 Conversion of Uroporphyrinogen III into Siroheme ...................................................................11 1.1.5 Diseases and Growth Defects Caused by Tetrapyrrole Deficiency .............................................12 1.2 Porphobilinogen Synthase ..................................................................................................................13 1.2.1 Alaremycin, an Antibiotic Structurally Related to 5-Aminolevulinic Acid.................................14 1.3 Uroporphyrinogen III Synthase ..........................................................................................................15 1.4 Protoporphyrinogen IX Oxidase .........................................................................................................17 1.4.1 Tobacco Protoporphyrinogen IX Oxidase ...................................................................................18 1.5 Heme Biosynthesis in Archaea ...........................................................................................................19 1.5.1 A Second Pathway for Heme Biosynthesis in Desulfovibrio vulgaris ........................................20 1.6 Aim of the Study.................................................................................................................................22 2 MATERIALS AND METHODS ...................................................................................... 23 2.1 Instruments and Chemicals .................................................................................................................23 2.1.1 Instruments ..................................................................................................................................23 2.1.2 Chemicals and Kits......................................................................................................................24 2.2 Strains and Plasmids ...........................................................................................................................25 2.3 Growth Media and Media Additives...................................................................................................27 2.3.1 Growth Media..............................................................................................................................27 2.3.2 Media Additives ..........................................................................................................................30 2.4 Microbiological Techniques ...............................................................................................................30 2.4.1 Sterilization..................................................................................................................................30 2.4.2 Cultivation of Bacteria and Archaea............................................................................................30 2.4.3 Determination of Cell Density.....................................................................................................31 2.4.4 Storage of Bacterial Strains .........................................................................................................31 2.4.5 Determination of Minimal Inhibitory Constants..........................................................................31 2.5 Molecular Biology Techniques...........................................................................................................32 2.5.1 Preparation of DNA.....................................................................................................................32 2.5.2 Determination of DNA Concentration.........................................................................................33 2.5.3 Transformation of Bacteria by the RbCl2 Method.......................................................................33 2.5.4 Electrophoretic Separation of DNA.............................................................................................34 2.5.5 Site-Directed Mutagenesis of DNA.............................................................................................35 2.5.6 Amplification of DNA Fragments by PCR..................................................................................36 2.5.7 Enzymatic Modification of DNA ................................................................................................37 2.6 Protein Biochemical Methods.............................................................................................................38 2.6.1 Production and Purification of Pseudomonas aeruginosa Porphobilinogen Synthase ................38 2.6.2 Production and Purification of Methanosarcina barkeri Porphobilinogen Synthase ..................40 2.6.3 Production and Purification of Arabidopsis thaliana Uroporphyrinogen III Synthase................41 2.6.4 Production and Purification Tobacco Protoporphyrinogen IX Oxidase ......................................43 2.6.5 Concentrating Protein Solutions..................................................................................................44 2.6.6 Determination of Protein Concentration......................................................................................44 2.6.7 Determination of FAD Content ...................................................................................................44
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