On the enzymatic mechanism of 4-hydroxybutyryl-CoA dehydratase and 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum [Elektronische Ressource] / vorgelegt von Jin Zhang

Zhang

Sujets

• Biologie

Informations

• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 15
• Poids de l'ouvrage: 6 Mo

Extrait

On the enzymatic mechanism of 4-hydroxybutyryl-CoA
dehydratase and 4-hydroxybutyrate CoA-transferase
from Clostridium aminobutyricum



Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)

dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von


Jin Zhang
aus Xi´an, V.R. China
Marburg/Lahn, Germany 2010
Die Untersuchungen zur vorliegenden Arbeit wurden von April 2006 bis März 2010 im
Laboratorium für Mikrobiologie, Fachbereich Biologie, der Philipps Universität Marburg
unter der Leitung von Prof. Dr. W. Buckel durchgeführt.







Vom Fachbereich Biologie
der Philipps-Universität Marburg
als Dissertation am 04. 2010 angenommen.




Erstgutachter: Prof. Dr. Wolfgang Buckel
Zweitgutachter: Prof. Dr. Lars-Oliver Essen

Tag der mündlichen Prüfung am .



Die im zeitlichen Rahmen dieser Dissertation erzielten Ergebnisse sind in folgenden
Publikation veröffentlicht:

Martins, B., Messerschmidt, A., Friedrich, P., Zhang, J. & Buckel, W. (2007)
4-Hydroxybutyryl-CoA Dehydratase.
In: Handbook of Metalloproteins Online Edition (A. Messerschmidt, ed.) John Wiley & Sons
Ltd., Sussex, UK. (Review)

Li, F., Hinderberger, J., Seedorf, H., Zhang, J., Buckel, W. & Thauer, R. K. (2008)
Coupled ferredoxin- and crotonyl-CoA reduction with NADH catalyzed by the butyryl-CoA
dehydrogenase/ETF complex from Clostridium kluyveri.
The Journal of Bacteriology. 190, 843-850.
Macieira,S.*, Zhang, J.*, Velarde, M., Buckel, W., Messerschmidt, A. (2009)
Cystal Structure of 4-hydroxybutyrate CoA-Transferase from Clostridium aminobutyricum.
Biological Chemistry. 390 (12): 1251-1263
(* contributed equally to this work)
Macieira,S.*, Zhang, J.*, Velarde, M., Buckel, W., Messerschmidt, A. (2010)
Cystal Structure of the complex between 4-hydroxybutyrate CoA-Transferase from
Clostridium aminobutyricum and butyryl.CoA.
The FEBS Journal. (in submitting)
(* contributed equally to this work)
Zhang, J., Friedrich, P., Martins, B., Kim, J., Buckel, W.(2010)
Mutations in the active centre of 4-hydroxybutyryl-CoA dehydratase from Clostridium
aminobutyricum. (in preparation)

Contents
Abbreviations ................................................................................................ 8
Zusammenfassung ........................................................................................ 9
Summary ...................................................................................................... 11
1. Introduction .......................................................................................... 13
1.1 Overview of anaerobic energy metabolism ...................................................... 13
1.2 Glutamate fermentation pathway in anaerobic bacteria ................................... 14
1.3 Clostridium aminobutyricum ............................................................................ 18
1.4 Fermentation of 4-aminobutyrate in C. aminobutyricum ................................. 19
1.5 4-Hydroxybutyrate CoA-transferase from ....................... 21
1.6 4-Hydroxybutyryl-CoA dehydratase from C. aminobutyricum23
1.7 Proposed mechanism of dehydration via a ketyl radical .................................. 26
1.8 Cofactors in 4-hydroxybutyryl-CoA dehydratase ............................................ 29
th1.9 4-Hydroxybutyryl-CoA dehydratase in the 5 CO -fixation pathway ............. 33 2
1.10 Goals of this work ............................................................................................ 35
2. Materials and Methods ........................................................................ 36
2.1 Materials ........................................................................................................... 36
2.1.1 Chemicals and reagents ............................................................................... 36
2.1.2 Instruments, gases and columns .................................................................. 36
2.1.3 Bacterial strains and cultures ...................................................................... 37
2.1.4 Plsmids ........................................................................................................ 38
2.1.5 Oligonucleotides ......................................................................................... 38
2.1.6 Media .......................................................................................................... 41
2.1.7 Antibiotics ................................................................................................... 41
4

2.1.8 Molecular biology kits ................................................................................ 42
2.2 Molecular Biological Methods ......................................................................... 43
2.2.1 Isolation of genomic DNA from C. aminobutyricum ................................. 43
2.2.2 Isolation of plasmid DNA ........................................................................... 44
2.2.3 Determination of DNA concentration and purity ....................................... 44
2.2.4 Agarose gel electrophoresis ........................................................................ 44
2.2.5 DNA extraction from agarose gel ............................................................... 45
2.2.6 DNA restriction and ligation ....................................................................... 45
2.2.7 Preparation of competent Escherichia coli cells for electrotransformation 46
2.2.8 Electrotransformation ................................................................................. 46
2.2.9 PCR reaction ............................................................................................... 46
2.2.10 Cloning of the genes ................................................................................... 48
2.2.11 Sequencing of the cloned genes .................................................................. 48
2.2.12 Site directed mutagenesis ............................................................................ 48
2.3 Biochemical methods ....................................................................................... 50
2.3.1 Gene expression in E. coli and protein purification .................................... 50
2.3.2 Purification of other proteins ...................................................................... 52
2.3.3 Determination of protein concentration ...................................................... 54
2.3.4 Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 54
2.3.5 Gel-filtration ............................................................................................... 56
2.3.6 Enzyme activity assays ............................................................................... 57
2.3.7 Iron protein reconstitution........................................................................... 59
2.3.8 Non-heme iron determination with Ferene ................................................. 59
2.3.9 Acid-labile sulfur determination ................................................................. 60
2.3.10 Flavin determination by UV-Vis ................................................................ 62
5

2.3.11 MALDI-TOF Mass Spectrometry............................................................... 62
2.3.12 EPR Spectroscopy ....................................................................................... 63
2.4 Chemical synthesis ........................................................................................... 63
2.4.1 Acetyl-CoA, butyryl-CoA and crotonyl-CoA synthesis by anhydride ....... 63
2.4.2 CoA-esters synthesis by 4-hydroxybutyryl-CoA transferase ..................... 64
2.4.3 4-Hydroxyvaleryl-CoA synthesis ............................................................... 64
3. Results .................................................................................................... 65
3.1 The recombinant 4-hydroxybutyryl-CoA dehydratase (AbfD) in E. coli ........ 65
3.1.1 Sequence analysis of abfD .......................................................................... 65
3.1.2 Cloning and expression of abfD in E.coli ................................................... 68
3.1.3 Purification of the recombinant AbfD ........................................................ 68
3.1.4 Physical and chemical characterization of the recombinant protein ........... 69
3.1.5 Vinylacetyl-CoA ∆-isomerase .................................................................... 77
3.1.6 Mutagenesis of recombinant AbfD ............................................................. 80
3.2 The recombinant 4-hydroxybutyrate CoA-transferase (AbfT) in E. coli ......... 85
3.2.1 Sequence analysis of AbfT ......................................................................... 85
3.2.2 Cloning and expression of abfT in E.coli .................................................... 87
3.2.3 Protein purification and analysis ................................................................. 90
3.2.4 Mutagenesis in the active site of recombina