The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 143 Bl. Ill., graph. Darst. |
Internformat
MARC
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| 003 | DE-604 | ||
| 005 | 20150408 | ||
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| 008 | 150123s2014 ad|| m||| 00||| eng d | ||
| 035 | |a (OCoLC)900871352 | ||
| 035 | |a (DE-599)GBV812621301 | ||
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| 041 | 0 | |a eng | |
| 049 | |a DE-12 |a DE-188 | ||
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| 100 | 1 | |a Hartmann, Tobias |e Verfasser |0 (DE-588)1063815754 |4 aut | |
| 245 | 1 | 0 | |a The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus |c von Tobias Hartmann |
| 264 | 1 | |c 2014 | |
| 300 | |a 143 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Potsdam, Univ., Diss., 2014 | ||
| 650 | 0 | 7 | |a Molybdänenzym |0 (DE-588)4348950-3 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Rhodobacter capsulatus |0 (DE-588)4263732-6 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Dehydrogenasen |0 (DE-588)4149014-9 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a Molybdänenzym |0 (DE-588)4348950-3 |D s |
| 689 | 0 | 1 | |a Dehydrogenasen |0 (DE-588)4149014-9 |D s |
| 689 | 0 | 2 | |a Rhodobacter capsulatus |0 (DE-588)4263732-6 |D s |
| 689 | 0 | |5 DE-604 | |
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-027725806 | ||
Datensatz im Suchindex
| _version_ | 1804152857938624512 |
|---|---|
| adam_text | TABLE OF CONTENTS
TABLE OF CONTENTS
ABBREVIATIONS
1
1. INTRODUCTION 5
1.1 MOLYBDOENZYMES 5
1.2 FORMATE DEHYDROGENASES 7
1.2.1 FDHS IN EUKARYOTES AND PROKARYOTES 7
1.2.2 MO/W-CONTAINING FDHS 11
1.2.2.1 CHAPERONES ARE
NECESSARY TO OBTAIN
ACTIVE FDH 16
1.2.2.2 THE
ACTIVE SITE AND
ITS PROPOSED CATALYTIC
MECHANISM 17
1.2.3 FDH FROM RHODOBACTER
CAPSULATUS 22
1.3 AIMS OF THIS
STUDY 24
2. MATERIALS AND METHODS
25
2.1 MATERIALS 25
2.1.1 CHEMICALS 25
2.1.2 BUFFERS AND
MEDIA 25
2.1.3 ANTIBIOTICS 25
2.1.4 MARKERS AND DYES 25
2.1.5 COMMERCIAL ENZYMES 25
2.1.6 DNA ISOLATION KITS 26
2.1.7 BACTERIAL STRAINS 26
2.1.8 OLIGONUCLEOTIDES 26
2.1.9 PLASMIDS 26
2.2 MOLECULAR BIOLOGICAL METHODS 27
2.2.1 CULTIVATION OF E.
COLI 27
2.2.2 COMPETENT E. COLI CELLS 27
2.2.3 TRANSFORMATION OF E. COLI 27
2.2.4 LONG TERM STORAGE OF E. COLI (GLYCEROL
STOCKS) 28
2.2.5 CLONING TECHNIQUES 28
2.2.5.1 POLYMERASE
CHAIN REACTION (PCR) 28
2.2.5.2 SITE DIRECTED
MUTAGENESIS 28
2.2.5.3 DNA RESTRICTION DIGESTION 29
2.2.5.4 DNA LIGATION 29
2.2.5.5 AGAROSE GEL
ELECTROPHORESIS 29
2.2.5.6 DNA ISOLATION FROM
AGAROSE GELS 29
2.2.5.7 DNA SEQUENCING 29
2.2.6 ISOLATION AND PURIFICATION
OF PLASMID DNA 29
2.3 BIOCHEMICAL METHODS 30
2.3.1 PROTEIN EXPRESSIONS 30
2.3.1.1 EXPRESSION OF RCFDH HETEROLOGOUS
IN E. COLI 30
2.3.1.2 EXPRESSION OF FDSGBA, FDSA AND
FDSC 30
2.3.1.3 EXPRESSION OF FDSGB
AND FDSD 30
2.3.2 OPTIMIZATION STUDIES ON PROTEIN EXPRESSION 30
2.3.3 PROTEIN PURIFICATIONS 31
2.3.3.1 PURIFICATION OF HETEROLOGOUSLY
EXPRESSED RCFDH 31
2.3.3.2 PURIFICATION OF FDSGBA, FDSGB AND FDSA 31
2.3.3.3 PURIFICATION OF FDSC
AND FDSD 31
2.3.4 BUFFER DESALTING USING
PD-10 COLUMNS 31
2.3.5 ULTRAFILTRATION 31
2.3.6 PHOTOMETRICAL DETERMINATION OF PROTEIN
CONCENTRATIONS 32
2.3.7 SDS-PAGE 32
HTTP://D-NB.INFO/1064680143
TABLE OF CONTENTS
2.4 ANALYTICAL METHODS 32
2.4.1 NATIVE PAGE 32
2.4.2 UV/VIS SPECTROSCOPY 32
2.4.3 ENZYME ASSAYS 33
2.4.3.1 FORMATE OXIDATION 33
2.4.3.2 C0
2
REDUCTION 33
2.4.3.3 DETECTION OF PRODUCED FORMATE 33
2.4.3.4 DIAPHORASE ACTIVITY 34
2.4.3.5 NITRATE REDUCTION 34
2.4.3.6 NITRITE
QUANTIFICATION 34
2.4.3.7 H
2
ASSAYS 34
2.4.4 FORM A AND FORM A-GMP
ANALYSIS WITH HPLC 35
2.4.5 5-GMP ANALYSIS WITH
HPLC 35
2.4.6 ICP-OES 35
2.4.7 FMN ANALYSIS AND
QUANTITATION 36
2.4.8 FMN RECONSTITUTION ASSAY 36
2.4.9 ANALYTICAL SIZE-EXCLUSION CHROMATOGRAPHY 36
2.4.10 SURFACE PLASMON
RESONANCE (BIACORE*) 36
2.4.11 THERMOFLUOR APPROACH 37
2.4.12 IN VITRO SULFURATION
ASSAY 37
2.4.13 SORBO ASSAY 37
3. RESULTS
38
3.1 DEVELOPMENT OF AN
EXPRESSION SYSTEM FOR RCFDH IN E. COLI 38
3.2 CHARACTERIZATION OF PURIFIED RCFDH 39
3.2.1 PROTEIN STABILITY AND OXYGEN
TOLERANCE 39
3.2.2 MOLECULAR WEIGHT AND QUATERNARY STRUCTURE
OF FDSGBA 41
3.2.3 UVA/IS SPECTROSCOPY OF RCFDH 42
3.2.4 DETERMINATION OF THE
COFACTORS BOUND
TO RCFDH 42
3.2.4.1 RELATIVE
METAL SATURATION 45
3.2.4.2 IDENTIFICATION OF THE MOLYBDENUM COFACTOR
BOUND TO RCFDH 45
3.2.4.3 DETECTION OF THE
FLAVIN COFACTOR BOUND
TO RCFDH 47
3.2.5 THE APO-FORM OF RCFDH 49
3.2.6 THE ALPHA SUBUNIT OF RCFDH 50
3.3 DETERMINATION OF THE CATALYTIC
CONSTANTS 51
3.3.1 OXIDATION OF FORMATE 51
3.3.1.1 PH AND TEMPERATURE
DEPENDENCY OF ENZYME ACTIVITY 51
3.3.1.2 KINETIC MECHANISM AND
PARAMETERS OF RCFDH 52
3.3.1.3 ACTIVITIES WITH
ARTIFICIAL ELECTRON
ACCEPTORS 53
3.3.1.4 INHIBITORS
OF RCFDH CATALYTIC
ACTIVITIES 54
3.3.2 REDUCTION OF C0
2
55
3.4 RCFDH SHOWS HYDROGENASE ACTIVITY 57
3.5 NAD
+
/NADH REGENERATION SYSTEM 58
3.5.1 FDSGB AS DIAPHORASE SUBUNIT 58
3.5.1.1 EXPRESSION OF THE GENES
FDSG AND FDSB 59
3.5.1.2 ANALYTICAL GEL
FILTRATION AND UVA/IS SPECTROSCOPY OF FDSGB 60
3.5.1.3 DIAPHORASE ACTIVITY
OF FDSGB 61
3.5.2 COMPARISON OF FDSGB AND
RCFDH DIAPHORASE ACTIVITIES 62
3.6 STUDIES ON THE MATURATION
OF RCFDH 63
3.6.1 EXPRESSION OF THE
GENES FDSC AND
FDSD 63
3.6.2 THE GENES
FDSC AND FDSD
ARE CODING FOR
MOLECULAR CHAPERONES 64
3.6.3 INFLUENCE OF FDSC AND FDSD
ON RCFDH OR FDSA ACTIVITY 66
3.6.4 PROTEIN-PROTEIN INTERACTION STUDIES ON RCFDH, FDSC, AND
FDSD 69
3.7 STUDIES ON THE
CATALYTIC MECHANISM OF RCFDH 71
TABLE OF CONTENTS
3.7.1 SITE DIRECTED
MUTAGENESIS OF AMINO ACIDS
AT THE
ACTIVE SITE OF FDSA 72
3.7.2 CHARACTERIZATION OF RCFDH VARIANTS 73
3.7.3 INFLUENCE OF RCFDH ACTIVE SITE
VARIANTS ON CATALYTIC ACTIVITY 76
3.8 RCFDH EXHIBITS NITRATE
REDUCTASE ACTIVITY 79
4. DISCUSSION 81
4.1 ROLE OF FDH
IN RHODOBACTER CAPSULATUS 81
4.2 PURIFICATION AND CHARACTERIZATION OF RCFDH 82
4.2.1 MATURATION OF ACTIVE
RCFDH DEPENDS ON THE PRESENCE
OF FDSC AND
FDSD... 85
4.2.2 RCFDH INTERCONVERTS
FORMATE AND C0
2
89
4.2.3 RCFDH IS ABLE TO USE NITRATE AND
H
2
AS SUBSTRATES 94
4.2.4 THE DIAPHORASE
SUBUNIT FDSGB HAS
NAD(H)-OXIDO-REDUCTASE ACTIVITY 97
4.3 RCFDH COORDINATES BIS-MGD, FMN
AND A COMPLEX FES CLUSTER
ELECTRON
TRANSPORT CHAIN 99
4.4 MOLYBDENUM AND TUNGSTEN
AS ACTIVE
SITE METAL IN FDHS 106
4.5 THE ROLE
OF SELENIUM IN THE
ACTIVE SITE
OF FDHS 108
4.6 THE CATALYTIC MECHANISM
OF MO/W BIS-MGD CONTAINING FDHS 109
4.6.1 A SULFUR LIGAND COMPLETES THE SIXTH
COORDINATION OF THE
MO/W BIS-MGD
IN
FDHS 109
4.6.2 THE ROLE
OF CONSERVED ACTIVE SITE
RESIDUES IN THE CATALYTIC MECHANISM
OF MO/W
BIS-MGD CONTAINING FDHS 110
4.6.3 THE PROPOSED CATALYTIC MECHANISM
OF RCFDH 112
4.7 OUTLOOK 116
REFERENCES 118
APPENDIX 131
FIGURES AND TABLES 137
ZUSAMMENFASSUNG 140
DANKSAGUNG 142
EIGENSTANDIGKEITSERKLARUNG 143
|
| any_adam_object | 1 |
| author | Hartmann, Tobias |
| author_GND | (DE-588)1063815754 |
| author_facet | Hartmann, Tobias |
| author_role | aut |
| author_sort | Hartmann, Tobias |
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| dewey-full | 572.79129385 |
| dewey-hundreds | 500 - Natural sciences and mathematics |
| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.79129385 |
| dewey-search | 572.79129385 |
| dewey-sort | 3572.79129385 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| indexdate | 2024-07-10T01:17:23Z |
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| language | English |
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| physical | 143 Bl. Ill., graph. Darst. |
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| spelling | Hartmann, Tobias Verfasser (DE-588)1063815754 aut The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus von Tobias Hartmann 2014 143 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Potsdam, Univ., Diss., 2014 Molybdänenzym (DE-588)4348950-3 gnd rswk-swf Rhodobacter capsulatus (DE-588)4263732-6 gnd rswk-swf Dehydrogenasen (DE-588)4149014-9 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Molybdänenzym (DE-588)4348950-3 s Dehydrogenasen (DE-588)4149014-9 s Rhodobacter capsulatus (DE-588)4263732-6 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027725806&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Hartmann, Tobias The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus Molybdänenzym (DE-588)4348950-3 gnd Rhodobacter capsulatus (DE-588)4263732-6 gnd Dehydrogenasen (DE-588)4149014-9 gnd |
| subject_GND | (DE-588)4348950-3 (DE-588)4263732-6 (DE-588)4149014-9 (DE-588)4113937-9 |
| title | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus |
| title_auth | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus |
| title_exact_search | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus |
| title_full | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus von Tobias Hartmann |
| title_fullStr | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus von Tobias Hartmann |
| title_full_unstemmed | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus von Tobias Hartmann |
| title_short | The Mo-containing and NAD + dependent formate dehydrogenase from Rhodobacter capsulatus |
| title_sort | the mo containing and nad dependent formate dehydrogenase from rhodobacter capsulatus |
| topic | Molybdänenzym (DE-588)4348950-3 gnd Rhodobacter capsulatus (DE-588)4263732-6 gnd Dehydrogenasen (DE-588)4149014-9 gnd |
| topic_facet | Molybdänenzym Rhodobacter capsulatus Dehydrogenasen Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027725806&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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