Enzymes of two clostridial amino-acid fermentation pathways [Elektronische Ressource] / vorgelegt von Gloria E. Herrmann T.

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Biologie

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Publié par	philipps-universitat_marburg
Publié le	01 janvier 2008
Nombre de lectures	28
Langue	Deutsch

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Enzymes of two clostridial amino-acid fermentation pathways

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

dem
Fachbereich Biologie
der Philipps-Universität Marburg
vorgelegt von

Gloria E. Herrmann T.
aus Maracaibo, Venezuela

Marburg/Lahn 2008
Die Untersuchungen zur vorliegenden Arbeit wurden von Januar 2001 bis Dezember 2005 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. Wolfgang Buckel
Zweitgutachter: HD. Dr. Thorsten Selmer

Tag der mündlichen Prüfung: _______________

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

Herrmann, G., Selmer, T., Jessen, H.J., Gokarn, R.R., Delifonova, O., Gort, S.J. & Buckel, W.
(2005) Two β-alanyl-CoA:ammonia lyases in Clostridium propionicum. FEBS J. 27, 813-21.

Boiangiu, C. D., Jayamani, E., Brugel, D., Herrmann, G., Kim, J., Forzi, L., Hedderich, R.,
Vgenopoulou, I., Pierik, A. J., Steuber, J. & Buckel, W. (2005) Sodium ion pumps and
hydrogen production in glutamate fermenting anaerobic bacteria. J. Mol. Microbiol.
Biotechnol. 10, 105-119.

Herrmann, G., Jayamani, E., Mai, G. & Buckel, W. (2008) Energy conservation via electron
transferring flavoprotein (ETF) in anaerobic bacteria. J. Bacteriol. 190, 784-791.

Heine, A., Herrmann, G., Selmer, G., Silber, K., Klebe, G., Buckel, W. & Reuter, K. (2008)
Cristallographic analysis of β-alanyl-CoA: Ammonia Lyase (Acl) from Clostridium
propionicum. In preparation.

To Ursula Beate Herrmann Twarz
and Emil Herrmann Belloso - 1 -
Table of contents
Abreviatons 4
Zusamenfasung 5
Sumary 6
Introduction 7
1. Energy metabolism of anaerobic bacteria 7
2. Fatty acid oxidation and oxidative phosphorylation 13
3. Butyryl-CoA dehydrogenase 15
4. The aim of this work 17
Materials and Methods 18
1. Bacterial growth 18
1.1. Anaerobic cultures of clostridia 18
1.2. Glutamate fermentation by Clostridium tetanomorphum 19
2. General biochemical and immunological methods 20
2.1. Determination of protein concentration 20
2.2. Synthesis, isolation and identification of CoA thioesters. MALDI probes 20
2.3. DTNB test for quantification of CoA and its derivates 21
2.4. SDS-PAGE 21
2.5. Blue native gel electrophoresis 22
2.6. Western Blot 23
2.7. Antibody production and immune serum screeinig 23
2.8. Affinity purification of specific IgG antibodies 24
3. Protein isolation and characterization 26
3.1. Purification of lactyl-CoA dehydratase from Clostridium propionicum 26
3.2. MALDI-TOF assay for lactyl-CoA dehydratase 26
3.3. β-alanyl-CoA ammonia lyase purification from C. propionicum 27
3.4. Superdex 200 size exclusion chromatography 27
3.5. Membrane preparation from C. tetanomorphum cell pellet 28
3.6. Partial isolation of membrane proteins 28
3.7. Purification of butyryl-CoA dehydrogenase-ETF from C. tetanomorphum 29
3.8. Superose 6 size exclusion chromatography 30
3.9. N-terminal sequencing of butyryl-CoA dehydrogenase-ETF subunits 30
3.10. Flavin cofactor investigation in butyryl-CoA dehydrogenase-ETF 30 - 2 -
3.10.1. Spectrophotometric flavin identification 30
3.10.2. Flavin quantification by HPLC 31
4. Enzyme assays 31
4.1. β-Alanyl-CoA ammonia lyase assay
4.2. Ammonia donors for β-alanyl-CoA ammonia lyase assay 31
4.3. Butyryl-CoA dehydrogenase-ETF aerobic assays 32
4.3.1. Ferricenium hexafluorophosphate assay 32
4.3.2. pH and buffer influence on butyryl-CoA dehydrogenase activity 32
4.3.3. Butyryl-CoA oxidation at 290 nm 32
4.3.4. NADH activity by INT 32
4.3.5. NADH consumption and peroxide formation in the ABTS test 33
4.4. Butyryl-CoA dehydrogenase anaerobic assays 33
4.4.1. Ferricenium hexafluorophophate assay in membrane fractions 33
4.4.2. Methylviologen assay 34
4.4.3. NADH/FAD crotonyl-CoA reduction assay 35
5. Immuno-gold labelling and electron microscopy techniques 36
Results 37
I. Butyryl-CoA dehydrogenase-ETF from Clostridium tetanomorphum 37
1. Purification of the butyryl-CoA dehydrogenase-ETF complex 37
2. N-terminal sequencing of butyryl-CoA dehydrogenase-ETF complex 38
3. Size and molecular composition of the butyryl-CoA dehydrogenase complex 40
4. Flavin determination in butyryl-CoA dehydrogenase-ETF complex 41
5. Enzyme assays 42
5.1. Aerobic catalysis of the complex butyryl-CoA dehydrogenase-ETF 42
5.1.1. Butyryl-CoA dehydrogenase activity by the ferricenium assay 42
5.1.2. pH and buffer influence on the BCD-ETF activity 45
5.1.3. Butyryl-CoA dependent oxidase activity at 290 nm 46
5.1.4. NADH oxidation by INT 46
5.1.5. Hydrogen peroxide formation in correlation with NADH oxidation 47
5.2. Anaerobic catalysis of the complex butyryl-CoA dehydrogenase-ETF 50
5.2.1. Methylviologen assay for crotonyl-CoA reductase activity 50
5.2.2. NADH/FAD crotonyl-CoA reductase assay 52
5.2.3. Butyryl-CoA dehydrogenase activity in membrane fractions 56
6. Immunological methods applied to butyryl-CoA dehydrogenase-ETF 57 - 3 -
6.1. Antibodies for butyryl-CoA dehydrogenase-ETF 57
6.2. Immuno-gold labelling and electron microscopy techniques 60
7. Cultures of C. tetanomorphum on glutamate and crotonate 63
II. Lactyl-CoA dehydratase and β-alanyl-CoA ammonia lyase from Clostridium
propionicum 66
1. Cultures of C. propionicum 66
1.1. Growth on alanine and threonine 66
1.2. Growth on cyclopropane carboxylic acid 67
2. Purification of lactyl-CoA dehydratase from C. propionicum 68
3. Purification and molecular characterization of β-alanyl-CoA ammonia lyase 71
4. β-Alanyl-CoA ammonia lyase activy 72
Discusion 76
1. Lactyl-CoA dehydratase and β-alanyl-CoA ammonia lyase from C. propionicum 76
2. Butyryl-CoA dehydrogenase-ETF from C. tetanomorphum 80
3. Butyryl-CoA dehydrogenase-ETF complex as a module for metabolic shift 85
Refrences 87

- 4 -
Abbreviations

DTT Dithiothreitol
DTNB 5,5'-dithiobis(2-nitrobenzoate)
ABTS 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid
INT Iodonitrotetrazolium chloride
FcPF Ferricenium hexafluorophosphate 6
Fd Ferredoxin
LCD Lactyl-CoA dehydratase
BCD Butyryl-CoA dehydrogenase
ETF Electron transfer flavoprotein
ACL β-Alanyl-CoA ammonia lyase
ACAD Acyl-CoA dehydrogenase
SCAD Short chain acyl-CoA dehydrogenase
MCAD Medium chain acyl-CoA dehydrogenase
LCAD Long chain acyl-CoA
VLCAD Very long chain acyl-CoA dehydrogenase
HAD 3-hydroxyacyl-CoA dehydrogenase
TBS Tris buffer saline
PBS Phosphate buffer saline
PBHS Phosphate buffer high saline
HRP Horseradish-peroxidase
PVDF Polyvinylidene fluoride
PMSF Phenylmethanesulphonylfluoride

- 5 -
Zusammenfassung
Zwei Enzyme der Alanin-Fermentation von Clostridium propionicum wurden biochemisch
untersucht. Das Enzym (R)-Lactyl-CoA Dehydratase, das eine chemisch schwierige
Wasserabspaltung von (R)-Lactyl-CoA zum Acrylyl-CoA katalysiert, konnte unter strikt
anaeroben Bedingungen partiell aufgereinigt werden. Grüne Fraktionen der Komponente D
und dunkle braune Fraktionen der Komponente A (Aktivator) wurden erhalten. Über MALDI
Massenspektrometrie konnte Lactyl-CoA (m/z = 840), das Hydratysierungsprodukt von
Acrylyl-CoA, nachgewiesen werden. Dazu wurde Acrylyl-CoA, Komponente D und
2+Komponente A