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Substratstereochemie Und Untersuchungen Zum Mechanismus Der 4-Hydroxybutyryl-Coa-Dehydratase Aus Clostridium Aminobutyricum

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Substratstereochemie Und Untersuchungen Zum Mechanismus Der 4-Hydroxybutyryl-Coa-Dehydratase Aus Clostridium Aminobutyricum Substratstereochemie und Untersuchungen zum Mechanismus der 4-Hydroxybutyryl-CoA-Dehydratase aus Clostridium aminobutyricum Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg vorgelegt von Peter Friedrich aus Marburg/Lahn Marburg/Lahn 2008 Die Untersuchungen zur vorliegenden Arbeit wurden von November 2003 bis November 2008 am Fachbereich Biologie der Philipps-Universität Marburg unter der Leitung von Herrn Prof. Dr. W. Buckel durchgeführt. Vom Fachbereich Biologie der Philipps-Universität Marburg als Dissertation am angenommen. Erstgutachter: Prof. Dr. W. Buckel Zweitgutachter: Prof. Dr. R. Thauer Tag der mündlichen Prüfung: 18.12.2008 für meine Familie für Christine Die Ergebnisse dieser Dissertation sind in folgenden Publikationen veröffentlicht: Friedrich P, Darley DJ, Golding BT, Buckel W (2008) The complete stereochemistry of the enzymatic dehydration of 4-hydroxybutyryl coenzyme A to crotonyl coenzyme A. Angew. Chem. Int. Ed. Engl. 47:3254-3257. Der stereochemische Verlauf der enzymatischen Wassereliminierung von 4-Hydroxybutyryl-Coenzym A zu Crotonyl- Coenzym A. Angew. Chem. 120:3298-3301 Martins BM, Messerschmidt A, Friedrich P, Zhang J, Buckel W (2007) 4-Hydroxybutyryl- CoA dehydratase. In: Messerschmidt A (ed) Handbook of Metalloproteins Online Edition. John Wiley & Sons Ltd., Sussex, UK. Publisched online Dez 2007 weitere Veröffentlichungen: Scott R, Näser U, Friedrich P, Selmer T, Buckel W, Golding BT (2004) Stereochemistry of hydrogen removal from the 'unactivated' C-3 position of 4-hydroxybutyryl-CoA catalysed by 4-hydroxybutyryl-CoA dehydratase. Chem. Commun. (Camb.):1210- 1211 1 Inhaltsverzeichnis Abkürzungsverzeichnis ....................................................................................... 6 Verzeichnis der in dieser Arbeit synthetisierten Verbindungen: ................... 7 Zusammenfassung ............................................................................................... 9 Summary ............................................................................................................ 11 1. Einleitung ....................................................................................................... 13 1.1 Glutamatabbau in der anaeroben Nahrungskette ........................................................... 13 1.2 Dehydratasen im anaeroben Stoffwechsel ..................................................................... 17 1.3 Funktionen und Vorkommen der 4-Hydroxybutyryl-CoA-Dehydratase ....................... 20 1.4 Aufbau, Struktur und Kinetik der 4-Hydroxybutyryl-CoA-Dehydratase ...................... 22 1.5 Mechanistische Modelle ................................................................................................. 23 1.5.1 Zwei Mechanismen der Butyryl-CoA-Dehydrogenase: über Hydridtransfer oder über ein Biradikal zwischen Substrat und FAD-Kofaktor ...................................... 23 1.5.2 Zwei Mechanismen der 4-Hydroxybutyryl-CoA-Dehydratase: über ein Ketylradikal oder mit Hydridtransfer über 4-Hydroxycrotonyl-CoA .......................................... 25 1.6 Zielsetzung ..................................................................................................................... 28 1.6.1 Stereoselektivität an C2 ........................................................................................... 28 1.6.2 Interaktion mit verschiedenen Substraten ............................................................... 28 1.6.3 Stereochemie an C4 ................................................................................................. 29 2 Material und Methoden ................................................................................. 30 2.1 Organismen .................................................................................................................... 30 2.2 Expressionskonstrukte .................................................................................................... 30 2.3 Medien für Anzucht von Organismen ............................................................................ 30 2.3.1 Anzucht von Clostridium aminobutyricum mit Selektivmedium ........................... 30 2.3.2 LB-Medium ............................................................................................................. 31 2.4 Allgemeine biochemische Analysen .............................................................................. 32 2.4.1 Eisenbestimmung mit Ferene .................................................................................. 32 2.4.2 Bestimmung von Proteinkonzentrationen ............................................................... 32 2.4.3 SDS-Polyacrylamid-Gelelektrophorese .................................................................. 33 2.5 Enzymreinigungen ......................................................................................................... 34 2.5.1 4-Hydroxybutyryl-CoA-Dehydratase aus Clostridium aminobutyricum ................ 34 2.5.2 4-Hydroxybutyrat-Dehydrogenase aus Clostridium aminobutyricum .................... 35 2 2.5.3 4-Hydroxybutyrat-CoA-Transferase aus Clostridium aminobutyricum ................. 36 2.5.4 Reinigung von Crotonase aus Rinderleber .............................................................. 38 2.5.5 Produktion und Reinigung der Malat-Synthase aus Escherichia coli ..................... 39 2.6 Enzymaktivitätsbestimmungen ...................................................................................... 41 2.6.1 4-Hydroxybutyryl-CoA-Dehydratase ...................................................................... 41 2.6.2 4-Hydroxybutyrat-Dehydrogenase .......................................................................... 42 2.6.3 4-Hydroxybutyrat-CoA-Transferase ....................................................................... 42 2.6.4 Crotonase ................................................................................................................. 43 2.6.5 Malat-Synthase ........................................................................................................ 43 2.6.6 Butyryl-CoA-Dehydrogenaseaktivität der 4-Hydroxybutyryl-CoA-Dehydratase .. 44 2.6.7 Aktivitätstest für die Rückreaktion der 4-Hydroxybutyryl-CoA-Dehydratase ....... 44 2.6.8 Aktivitätsassay der 4-Hydroxybutyryl-CoA-Dehydratase bei 260 - 290 nm .......... 45 2.7 Substratbestimmungen ................................................................................................... 46 2.7.1 Acetat ...................................................................................................................... 46 2.7.2 4-Hydroxybutyrat .................................................................................................... 47 2.7.3 Malat ........................................................................................................................ 48 2.8 Substratsynthesen ........................................................................................................... 48 2.8.1 Acetyl-CoA (1), Butyryl-CoA (2) und Crotonyl-CoA (3) über Anhydride ............ 48 2.8.2 CoA-Ester-Synthesen über 4-Hydroxybutyrat-CoA-Transferase ........................... 49 2.8.2.1 4-Hydroxybutyryl-CoA (4) .................................................................................. 49 2.8.2.2 Butyryl-CoA (5) ................................................................................................... 49 2.8.3 Herstellung und Analyse einer Markierung am C2 von Butyryl-CoA über Crotonase ................................................................................................................. 50 2.9 Inhibitorensynthesen ...................................................................................................... 51 2.9.1 Acetoacetyl-CoA (6) ............................................................................................... 51 2.9.2 3-Hydroxybutyryl-CoA (7) ..................................................................................... 52 2.9.3 1-Iodo-4-butanol (8) ................................................................................................ 52 2.9.4 4-Hydroxybutan-CoA-Ether (9) .............................................................................. 52 2.10 Analysen zur Stereoselektivität der 4-Hydroxybutyryl-CoA-Dehydratase am C2 ...... 53 2.10.1 Reaktion von 4-Hydroxybutyryl-CoA zu Crotonyl-CoA ...................................... 53 2 2.10.2 Reaktion mit (S)-[2- H1]Butyryl-CoA .................................................................. 53 2 2.10.3 (R)-[2- H1]Butyryl-CoA (R-5) oder Reaktion mit Butyryl-CoA .......................... 54 2.11 Ammonium-Lyaseaktivität der Dehydratase ............................................................... 54 2.12 MALDI-TOF-Massenspektrometrie ............................................................................ 55 3 2.13 Mössbauer-Spektroskopie – Effekte der Substratbindung ........................................... 55 2.14 EPR-Spektroskopie – Effekte verschiedener Substrate ............................................... 57 2.15 ENDOR-Spektrokopie – Effekte der Substratbindung ................................................ 58 2.16 NMR-Charakterisierung von Syntheseprodukten ........................................................ 59 2.17 NMR-Experimente über chirale Verschiebung ............................................................ 59 2.17.1 Europium Tris[3-(Heptafluoro-Propylhydroxymethylene)-(+)-Kampfer]-Ligand
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