Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation: = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis Book |
| Language: | English |
| Published: |
Würzburg
September 2013
|
| Subjects: | |
| Online Access: | Volltext Inhaltsverzeichnis |
| Item Description: | veröffentlicht: 2017 |
| Physical Description: | xiii, 128 Seiten Illustrationen, Diagramme |
Staff View
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| 245 | 1 | 0 | |a Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation |b = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |c submitted by Shambhavi Mishra from Kanpur |
| 246 | 1 | 1 | |a Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |
| 264 | 1 | |a Würzburg |c September 2013 | |
| 300 | |a xiii, 128 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 500 | |a veröffentlicht: 2017 | ||
| 502 | |b Dissertation |c Julius-Maximilians-Universität Würzburg |d 2014 | ||
| 546 | |a Zusammenfassung in deutscher und englischer Sprache | ||
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| 650 | 0 | 7 | |a Benzoate |0 (DE-588)4336535-8 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a Rhodopseudomonas palustris |0 (DE-588)4356032-5 |2 gnd |9 rswk-swf |
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Record in the Search Index
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| adam_text | STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE
ENZYMES INVOLVED IN THE MENAQUINONE BIOSYNTHESIS
AND BENZOATE DEGRADATION
STRUKTURELLE UND FUNKTIONELLE CHARAKTERISIERUNG VON ENZYMEN,
DIE AN DER MENAQUINON-BIOSYNTHESE UND DER BIODEGRADATION VON BENZOAT
BETEILIGT SIND
DOCTORAL THESIS FOR A DOCTORAL DEGREE
AT THE GRADUATE SCHOOL OF LIFE SCIENCES,
JULIUS-MAXIMILIANS-UNIVERSITAET WUERZBURG,
SECTION BIOMEDICINE
SUBMITTED BY
SHAMBHAVI MISHRA
FROM
KANPUR
WUERZBURG
2013
LIST OF TABLES
LIST OF
FIGURES............................................................................................................II
LIST OF A B B RE V IA TIO N S
..............................................................................................V
S UM M
ARY................................................................................................................
VIII
ZUSAM M
ENFASSUNG................................................................................................XI
CHAPTER
1.................................................................................................................
1
STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF BADL FROM
RHODOPSEUDOMONAS
PALUSTRIS...........................................................................1
1.
INTRODUCTION.........................................................................................................3
1.1 AROMATIC COMPOUNDS: AN INTRODUCTION
........................................................................
3
1.1.1 STABILITY OF AROMATIC COMPOUNDS
......................................................................
4
1.1.2 AROMATICS AND BIOGEOCHEMICAL CYCLES
.............................................................
4
1.2 MICROBIAL DEGRADATION OF AROMATIC
COMPOUNDS...........................................................4
1.2.1 AEROBIC AND ANAEROBIC MICROBIAL DEGRADATION
.................................................
5
1.2.2 CENTRAL BENZOIC ACID BIODEGRADATION
................................................................
8
1.3 BADL, A 2-KETOCYCLOHEXANECARBOXYL-COA
HYDROLASE....................................................10
1.3.1 BADL: A MEMBER OF THE CROTONASE SUPERFAMILY
...............................................
10
1.3.2 BADL AND MENB: INTRIGUING SIMILARITIES
............................................................
13
1.4
OBJECTIVES...................................................................................................................
14
2. MATERIALS AND
METHODS...................................................................................
16
2.1
MATERIALS.....................................................................................................................
16
2.1.1 PLASMID
...........................................................................................................
16
2.1.2 BACTERIAL STRAINS
..............................................................................................
16
2.1.3 LIGANDS
...........................................................................................................
16
2.1.4 CRYSTALLIZATION SCREENS
...................................................................................
16
2.1.5 LOW MOLECULAR WEIGHT CHEMICALS
..................................................................
17
2.1.6 SOLUTIONS
.........................................................................................................
18
2.1.7 INSTRUMENTS
......................................................................................................
19
2.1.8 CONSUMABLES
..................................................................................................
20
2.1.9 KITS
..................................................................................................................
20
2.1.10 SOFTWARE AND DATABASES
...........................
20
2.2 BACTERIAL
TRANSFORMATION............................................................................................
22
2.3 OVER-EXPRESSION OF RECOMBINANT
PROTEIN...................................................................
22
2.4 PROTEIN
PURIFICATION....................................................................................................23
2.4.1 ANION EXCHANGE CHROMATOGRAPHY: Q-SEPHAROSE
...........................................
23
2.4.2 SIZE EXCLUSION CHROMATOGRAPHY
.....................................................................
24
2.5 PROTEIN
CHARACTERIZATION.............................................................................................25
2.5.1 SDS-PAGE
.....................................................................................................
25
2.5.2 PROTEIN CONCENTRATION DETERMINATION
.............................................................
26
2.5.3 ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY
.....................................................
27
2.5.4 ANALYTICAL ULTRACENTRIFUGATION
..........................................................................27
2.5.5 ATOMIC FORCE MICROSCOPY
................................................................................
28
2.5.6 DYNAMIC LIGHT SCATTERING
.................................................................................
28
2.6 CRYSTALLIZATION METHODS
.............................................................................................
29
2.6.1 FREEZING AND DIFFRACTION EXPERIMENTS
.............................................................
30
2.1 DATA COLLECTION AND STRUCTURE SOLUTION METHODS
......................................................
31
2.7.1 DATA PROCESSING
.............................................................................................
32
2.7.2 MOLECULAR REPLACEMENT
...................................................................................
34
2.8 MODEL BUILDING AND STRUCTURE
REFINEMENT..................................................................35
2.8
.7
LIGAND AND HETERO COMPOUNDS GENERATION
....................................................
37
2.9 LIGAND
DOCKING............................................................................................................37
3. RESULTS AND
DISCUSSION..................................................................................
39
3.1 PROTEIN
PURIFICATION....................................................................................................39
3.2 PROTEIN
CHARACTERIZATION.............................................................................................40
3.3 PROTEIN
CRYSTALLIZATION................................................................................................41
3.4 STRUCTURE
DETERMINATION............................................................................................43
3.4.1 OVERALL STRUCTURE
.............................................................................................
45
3.4.2 THE MONOMER.
.................................................................................................
45
3.5
OLIGOMERIZATION..........................................................................................................49
3.5.1 THE TRIMER
......................................................................................................
50
3.5.2 THE HEXAMER
..................................................................................................
52
3.7
THE ACTIVE SITE TRIAD CREATED BY S138, D140 AND Y235
.....................................
57
3.8 ENZYME CATALYSIS AND COMPARISON WITH
MENB............................................................. 62
3.9
CONCLUSIONS................................................................................................................
65
CHAPTER I I
...............................................................................................................
67
STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF STAPHYLOCOCCAL MEND.. 67
4.
INTRODUCTION.......................................................................................................69
4.1 STAPHYLOCOCCUS AUREUS: A DISEASE-CAUSING AGENT
.......................................................
69
4.2 TREATMENT AND ASSOCIATED MORBIDITY AND M ORTALITY
..................................................
70
4.3 DEFENSE MECHANISM OF STAPHYLOCOCCUS
AUREUS..........................................................73
4.4 SMALL COLONY
VARIANTS.................................................................................................
76
4.5 THE SCVS: CAUSE OF PERSISTENT, RECURRENT AND ANTIBIOTIC-RESISTANT
INFECTIONS
...........
79
4.6
QUINONE......................................................................................................................
81
4.6.1 THE MENAQUINONE BIOSYNTHESIS PATHWAY
.......................................................
83
4.7
MEND..........................................................................................................................
86
4.8 MENAQUINONES IN
HUMAN............................................................................................88
4.9 SCV: AN ALTERNATIVE
TREATMENT....................................................................................
88
4.10
OBJECTIVES.................................................................................................................89
5. MATERIALS AND
METHODS...................................................................................90
5.1
MATERIALS.....................................................................................................................90
5.1.1 PLASMID
...........................................................................................................
90
5.1.2 BACTERIAL STRAINS
..............................................................................................
90
5.1.3 LIGANDS
...........................................................................................................
90
5.2 BACTERIAL
TRANSFORMATION............................................................................................90
5.3 PROTEIN
OVEREXPRESSION..............................................................................................
91
5.3.1 OVEREXPRESSION OF THE NATIVE PROTEIN
.............................................................
91
5.3.2 OVER-EXPRESSION OF SELENOMETHIONINE-SUBSTITUTED (SEMET) PROTEIN
...............
92
5.4 PROTEIN
PURIFICATION....................................................................................................92
5.4.1 AFFINITY CHROMATOGRAPHY
..................................................................................
92
5.4.2 SIZE EXCLUSION CHROMATOGRAPHY
.....................................................................
93
5.5 PROTEIN CONCENTRATION
DETERMINATION........................................................................94
5.6 CRYSTALLIZATION
METHODS.............................................................................................
95
5.6.1 MICROSEED MATRIX SEEDING
.............................................................................
95
5.7 DATA COLLECTION, PROCESSING AND STRUCTURE DETERMINATION
.........................................
95
6. RESULTS AND
DISCUSSION....................................................................................97
6.1 AFFINITY PURIFICATION OF NATIVE AND SELENOMETHIONINE SUBSTITUTED
MEND
...................
97
6.2 SIZE EXCLUSION
CHROMATOGRAPHY.................................................................................
97
6.2 MEND CRYSTALLIZATION (NATIVE AND
SELENOMETHIONINE)................................................99
6.3 STRUCTURE
DETERMINATION...........................................................................................101
6.3.1 OVERALL STRUCTURE
............................................................................................
101
6.3.2 OLIGOMERIZATION
.............................................................................................
104
6.4 COMPARISON OF SAMEND WITH THE KNOWN MEND
STRUCTURES.......................................109
6.4.1 LOOP STABILIZATION
..........................................................................................
112
6.4.2 CONFORMATION OF THE C-TERMINAL HELIX
............................................................
114
6.5 THE ACTIVE
SITE..........................................................................................................
114
6.5.1 THE THDP BINDING SITE
..................................................................................
115
6.5.2 DIVALENT METAL ION BINDING SITE
......................................................................
118
6.5.3. THE A- KG BINDING SITE
.................................................................................
119
6.5.4 THE ISOCHORISMATE BINDING SITE
...................................................................
120
6.6 BIOCHEMICAL
CHARACTERIZATION...................................................................................
120
6.6.
1 1DENTIFICATION OF THE REACTION PRODUCT
............................................................
121
6.6.2 ENZYME KINETICS
............................................................................................
121
6.6.3 MUTAGENESIS STUDIES
.....................................................................................
123
6.7 PROPOSED FUNCTION OF THE ANALYZED
RESIDUES.............................................................124
6.8
CONCLUSIONS..............................................................................................................
127
7.
REFERENCES..............................................................................................
129
ACKNOWLEDGMENTS
|
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| spelling | Mishra, Shambhavi 1978- Verfasser (DE-588)1123535817 aut Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind submitted by Shambhavi Mishra from Kanpur Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind Würzburg September 2013 xiii, 128 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier veröffentlicht: 2017 Dissertation Julius-Maximilians-Universität Würzburg 2014 Zusammenfassung in deutscher und englischer Sprache Staphylococcus aureus (DE-588)4182912-8 gnd rswk-swf Enzym (DE-588)4014988-2 gnd rswk-swf Biologischer Abbau (DE-588)4145625-7 gnd rswk-swf Benzoate (DE-588)4336535-8 gnd rswk-swf Rhodopseudomonas palustris (DE-588)4356032-5 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Benzoate (DE-588)4336535-8 s Biologischer Abbau (DE-588)4145625-7 s Enzym (DE-588)4014988-2 s Rhodopseudomonas palustris (DE-588)4356032-5 s Staphylococcus aureus (DE-588)4182912-8 s DE-604 Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:20-opus-90848 https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-90848 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029407834&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Mishra, Shambhavi 1978- Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind Staphylococcus aureus (DE-588)4182912-8 gnd Enzym (DE-588)4014988-2 gnd Biologischer Abbau (DE-588)4145625-7 gnd Benzoate (DE-588)4336535-8 gnd Rhodopseudomonas palustris (DE-588)4356032-5 gnd |
| subject_GND | (DE-588)4182912-8 (DE-588)4014988-2 (DE-588)4145625-7 (DE-588)4336535-8 (DE-588)4356032-5 (DE-588)4113937-9 |
| title | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |
| title_alt | Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |
| title_auth | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |
| title_exact_search | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |
| title_full | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind submitted by Shambhavi Mishra from Kanpur |
| title_fullStr | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind submitted by Shambhavi Mishra from Kanpur |
| title_full_unstemmed | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind submitted by Shambhavi Mishra from Kanpur |
| title_short | Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation |
| title_sort | structural and functional characterization of the enzymes involved in the menaquinone biosynthesis and benzoate degradation strukturelle und funktionelle charakterisierung von enzymen die an der menaquinon biosynthese und der biodegradation von benzoat beteiligt sind |
| title_sub | = Strukturelle und funktionelle Charakterisierung von Enzymen, die an der Menaquinon-Biosynthese und der Biodegradation von Benzoat beteiligt sind |
| topic | Staphylococcus aureus (DE-588)4182912-8 gnd Enzym (DE-588)4014988-2 gnd Biologischer Abbau (DE-588)4145625-7 gnd Benzoate (DE-588)4336535-8 gnd Rhodopseudomonas palustris (DE-588)4356032-5 gnd |
| topic_facet | Staphylococcus aureus Enzym Biologischer Abbau Benzoate Rhodopseudomonas palustris Hochschulschrift |
| url | https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-90848 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=029407834&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT mishrashambhavi structuralandfunctionalcharacterizationoftheenzymesinvolvedinthemenaquinonebiosynthesisandbenzoatedegradationstrukturelleundfunktionellecharakterisierungvonenzymendieandermenaquinonbiosyntheseundderbiodegradationvonbenzoatbeteiligtsind AT mishrashambhavi strukturelleundfunktionellecharakterisierungvonenzymendieandermenaquinonbiosyntheseundderbiodegradationvonbenzoatbeteiligtsind |