(R)-Indolelactyl-CoA dehydratase, the key enzyme of tryptophan reduction to indolepropionate in Clostridium sporogenes Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. Nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Diplom-Chemikerin Huan Li aus JiLin VR. China Marburg/Lahn, 2014 Die Untersuchungen zur vorliegenden Arbeit wurden von September 2010 bis Dezember 2013 im Max-Planck-Institut für terrestrische Mikrobiologie, Marburg und im Laboratorium für Mikrobiologie, Fachbereich Biologie, der Philipps-Universität Marburg (Hochschulkennziffer: 1180) unter der Leitung von Prof. Dr. Wolfgang Buckel durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am angenommen. Erstgutachter: Prof. Dr. Wolfgang Buckel Zweitgutachter: Prof. Dr. Johann Heider Tag der mündlichen Prüfung am: Für den gläubigen Menschen steht Gott am Anfang, für den Wissenschaftler am Ende aller seiner Überlegungen. Max Planck 献给最亲爱的爸爸妈妈 Index Zusammenfassung 1 Summary 2 Introduction 3 1. The role of gastrointestinal microbiota metabolites ........................................................... 3 2. Fermentation of amino acids and Stickland-reaction ......................................................... 5 3. Clostridium sporogenes ........................................................................................................... 7 4. 2-Hydroxyacyl-CoA dehydratases and the unusual radical H2O-elimination ................. 9 5. Family Ш CoA-transferases ................................................................................................ 15 6. Aim of thesis .......................................................................................................................... 17 Materials and methods 18 1. Chemicals, biochemichals and reagents ............................................................................. 18 1.1 Acyllactyl-CoA and acylacryl-CoA synthesis .............................................................. 18 1.2 Instruments, gases and columns ................................................................................... 18 1.3 Anaerobic work ............................................................................................................ 19 2. Microbiological methods ..................................................................................................... 19 2.1 Bacterial strains and cultures ........................................................................................ 19 2.2 Plasmid ......................................................................................................................... 20 2.3 Antibiotics ..................................................................................................................... 21 2.3 Genomic DNA isolation from C. sporogenes ............................................................... 21 2.4 Plasmid DNA isolation ................................................................................................. 22 2.5 Agarose gel electrophoresis .......................................................................................... 22 2.6 DNA extraction from agarose gels ............................................................................... 22 2.7 DNA restriction and ligation ........................................................................................ 22 2.8 Dialysis of ligation mixtures ......................................................................................... 23 2.9 Electrotransformation ................................................................................................... 23 2.10 Determination of DNA concentration and purity ......................................................... 23 2.11 Preparation of competent E. coli cells for electro transformation ................................ 23 2.12 PCR primers .................................................................................................................. 23 2.13 PCR reactions ............................................................................................................... 24 2.14 Cloning of the genes ..................................................................................................... 25 2.15 Sequencing of the cloned genes .................................................................................... 25 3. Biochemical methods ........................................................................................................... 25 3.1 End product and intermediates determination via ESI-Mass spectrometry and NMR . 25 3.2 Protein Purification ....................................................................................................... 25 3.2.1 Methods of cell disruption ....................................................................................... 25 3.2.2 Native protein purification of aryllactyl-CoA dehydratase from C.sporogenes grown on tryptophan or phenylalanine .................................................................... 26 3.2.3 Separation of CoA-transferase from the aryllactyl-CoA dehydatase complex ........ 27 3.2.4 Gene expression and purification of FldBC ............................................................. 27 3.2.5 Purification of the recombinant activator HgdC of 2-hydroxyglutaryl-CoA dehydratase of Acidaminococcus fermentans .......................................................... 28 3.2.6 Preparation of C. sporogenes membranes ................................................................ 28 3.3 Determination of the protein concentration .................................................................. 28 3.4 Polyacrylamide gel electrophoresis (PAGE) ................................................................ 29 3.5 Iron determination ........................................................................................................ 30 3.6 MALDI-TOF mass spectrometry ................................................................................. 31 3.7 Enzyme activity assays ................................................................................................. 32 3.7.1 Aryllactyl-CoA dehydratase .................................................................................... 32 3.7.2 CoA-transferase ....................................................................................................... 32 3.7.3 Tryptophan transaminase ......................................................................................... 33 3.7.4 Indoleacrylate reductase ........................................................................................... 33 3.7.5 Indolelactate dehydrogenase .................................................................................... 34 3.7.6 Rnf with ferricyanide ............................................................................................... 34 Results 35 1. Fermentations of phenylalanine and tryptophan in Clostridum sporogenes ................... 35 1.1 Growth of C. sporogenes on tryptophan or phenylalanine ........................................... 35 1.2 Intermediates and end products .................................................................................... 38 2. Studies on Aryllactyl-CoA dehydratase from C. sporogenes ........................................... 45 2.1 Chemical synthesis of CoA-thioesters .......................................................................... 45 2.2 Purification of the recombinant activator of the dehydratase ....................................... 47 2.3 Purifications of indolelactyl-CoA dehydratase and phenyllactyl-CoA dehydratase .... 48 2.3.1 The purification of indolelactyl-CoA dehydratase ................................................... 50 2.3.2 The purification of phenyllactyl-CoA dehydratase .................................................. 53 2.4 Enzyme activities of aryllactyl-CoA dehydratase ........................................................ 55 2.5 Purification of phenyllactyl-CoA with protease inhibitor and native gel ..................... 59 2.6 Time and temperature influence ................................................................................... 59 2.7 Complex fixing with AlF4 ............................................................................................ 60 2.8 Cloning and expression of fldBC in E. coli .................................................................. 61 2.9 Fe-determination ........................................................................................................... 65 2.10 Reduction with EuCl2 -DTPA complex as electron donor: Radical dehydration with an artificial electron donor EuCl2 ...................................................................................... 67 2.11 Sequence analysis and comparison ............................................................................... 68 2. Tryptophan transaminase, indolelactate dehydrogenase indoleacrylate reductatse and Rnf 70 3. Arginine fermentation in C. sporogenes, a probable oxidation partner of tryptophan in a Stickland reaction .............................................................................................................. 71 Discussion 72 1. The pathway of tryptophan fermentation in C. sporogenes ............................................. 72 2. Aryllactyl-CoA dehydratase complex ................................................................................ 74 3. Posttranslational modification of FldB .............................................................................