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Structure and Function of the Tungsten-Containing Active Site of Class II Benzoyl-Coa Reductases

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Structure and Function of the Tungsten-Containing Active Site of Class II Benzoyl-Coa Reductases Structure and function of the tungsten-containing active site of class II benzoyl-CoA reductases Inaugural-Dissertation zur Erlangung der Doktorwürde (Doctor rerum naturalium) Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau (D) vorgelegt von Simona G. Huwiler Freiburg im Breisgau (D), Dezember 2015 This thesis was conducted from November 2010 to November 2011 at the Institute of Biochemistry at Universität Leipzig (D) and from December 2011 to December 2015 at the Institute of Biology II (microbiology) at Albert-Ludwigs-Universität Freiburg i. Br. (D) in the group of Prof. Matthias Boll. Dekan der Fakultät für Biologie: Prof. Dr. Wolfgang Driever Promotionsvorsitzender: Prof. Dr. Stefan Rotter Betreuer der Arbeit: Prof. Dr. Matthias Boll Referent: Prof. Dr. Matthias Boll Koreferent: PD Dr. Ivan Berg Drittprüferin: Prof. Dr. Carola Hunte Datum der mündlichen Prüfung: 31.03.2016 Est autem admiratio desiderium quoddam sciendi, quod in homine contingit ex hoc quod vident effectum et ignorat causam, vel ex hoc quod causa talis effectus excedit cognitionem aut facultatem ipsius. Et ideo admiratio est causa delectationis, inquantum habet adjunctam spem consequendi cognitionem ejus quod scire desiderat. Thomas of Aquin (1225-1274)1 Now ‘wondering’ means ‘wanting to know something’: it is aroused when a man sees an effect and does not know its cause, or when he does not know or cannot understand how this cause could have that effect. Wondering therefore can cause him pleasure when it carries with it a real prospect of finding out what he wants to know.2 1 Summa theologiæ 1a2æ, 32,8 2 of Aquin, T. (1975) Summa theologiæ (Latin text, English translation, introduction, notes and glossary). d`Arcy, E. (ed). London: Blackfriars, pp. 1-172. Publication Publication Parts of this work have been published in the following article: Weinert, T.*, Huwiler, S.G.*, Kung, J.W., Weidenweber, S., Hellwig, P., Stärk, H.J., Biskup, T., Weber, S., Cotelesage J.J.H., George, G.N., Ermler, U., and Boll, M. (2015) Structural basis of enzymatic benzene ring reduction. Nat Chem Biol 11: 586-591. * These authors contributed equally to the work It is intended to publish further parts of this thesis in the future. Poster at the following conferences have presented this thesis to some extent: Huwiler, S.G., Weinert, T., Biskup, T., Kung, J.W., Hellwig, P., George, G., Stärk, H.J., Weber S., Ermler, U., and Boll, M. (2015) Structure and function of tungsten-containing class II benzoyl-coenzyme A reductase Annual conference of VAAM (Vereinigung für allgemeine und angewandte Mikrobiologie), 1. - 4. March, Marburg, D. Löffler, C., Huwiler, S., Anselmann, S.E.L., and Boll, M. (2014) Structure and function of the class II benzoyl-coenzyme A reductase from strictly anaerobic bacteria. Annual conference of VAAM, 5.-8. October, Dresden, D. Huwiler, S., Weinert, T., Ermler, U., Zelder, F., Männel-Croisé, Ch., George, G., Kung, J., Löffler, C., and Boll, M. (2013) The active site of the W-/Se-containing class II benzoyl-coenzyme A reductase complex in Geobacter metallireducens. Molybdenum and tungsten enzymes conference, 16. - 19. July, Sintra, PT. Huwiler, S., Löffler, C., Seifert, J., Stärk, H.J., George, G., Weinert, T., Ermler, U., Kung, J., and Boll, M. (2013) The (active site of the) W-/Se-containing class II benzoyl-coenzyme A reductase complex in Geobacter metallireducens. Annual conference of VAAM, 10.-13. March, Bremen, DE and 71th Annual congress of the Swiss Society for microbiology (SGM), 26.-27. June, Interlaken, CH. Huwiler, S.G., Oberender, J., Kung, J.W., and Boll, M. (2012) An expression system for the W-containing class II benzoyl-coenzyme A reductases in Geobacter metallireducens. Annual conference of VAAM, 18.- 21. March, Tübingen, D. Kung, J., Weinert, T., Dörner, K., Ermler, U., Friedrich, T., Huwiler, S., and Boll, M. (2011) Spectroscopic and structural studies of the active site components of tungsten-containing benzoyl-CoA reductase. Molybdenum and tungsten enzymes conference, 3.- 5. August, Edmonton, CAN. I Table of Contents Table of Contents Publication...................................................................................................................................................... I Table of Contents ......................................................................................................................................... II Summary .................................................................................................................................................... VIII Zusammenfassung .................................................................................................................................... XII Abbreviations and Terms ........................................................................................................................ XVI Introduction .................................................................................................................................................. 1 1. Definition, properties and occurrence of aromatic compounds ................................................................ 1 2. Aerobic degradation of aromatic compounds .................................................................................................. 2 3. Anaerobic degradation of aromatic compounds ............................................................................................. 3 3.1 Historical background and central intermediates .................................................................................. 3 3.2 Peripheral pathways leading to benzoyl-CoA .......................................................................................... 4 3.3 The benzoyl-CoA degradation pathway ..................................................................................................... 6 4. Benzoate-induced gene clusters in G. metallireducens…… ........................................................................... 9 5. Distribution of BCRs and energetic considerations….. .................................................................................... 9 6. The Birch reduction as a model for benzoyl-CoA reduction ..................................................................... 10 7. Class I BCRs ................................................................................................................................................................... 12 7.1 Molecular properties and electron-delivering system ....................................................................... 12 7.2 Reaction mechanism ....................................................................................................................................... 13 7.2.1 ATP-dependent electron transfer ................................................................................................. 13 7.2.2 Birch reaction-like mechanism ....................................................................................................... 14 8. Class II BCRs .................................................................................................................................................................. 15 8.1 Homology of class II BCR complex ............................................................................................................ 15 8.2 Molecular properties of class II BCR complex ....................................................................................... 16 8.2.1 Experimental composition ............................................................................................................... 16 8.2.2 Activities and electron-delivering system .................................................................................. 17 8.3 Structure and function of the active site of class II BCR ................................................................... 18 8.3.1 Molecular properties of active-site subunits BamBC of G. metallireducens................. 18 8.3.2 Activities of active-site subunits BamBC of G. metallireducens ......................................... 19 II Table of Contents 8.3.3 Crystal structure of active-site subunits BamBC of G. metallireducens .......................... 19 9. Aim of this thesis ........................................................................................................................................................ 21 Materials and Methods .............................................................................................................................. 2 3 1. General materials and methods ............................................................................................................................ 23 1.1 Chemicals and gases ....................................................................................................................................... 23 1.2 Synthesis of CoA-esters ................................................................................................................................. 23 1.2.1 Benzoyl-CoA .......................................................................................................................................... 23 1.2.2 1,5-Dienoyl-CoA ................................................................................................................................... 24 1.2.3 1-Monoenoyl-CoA .............................................................................................................................. 26 1.2.4 CoA
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