Structural and functional studies of tRNA-guanine transglycosylase [Elektronische Ressource] : a putative drug target for shigellosis therapy / vorgelegt von Bernhard Stengl

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• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2006
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

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Structural and Functional Studies of
tRNA-Guanine Transglycosylase:
A putative Drug Target for Shigellosis Therapy




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


dem
Fachbereich Pharmazie
der PHILIPPS-UNIVERSITÄT MARBURG
vorgelegt von



Bernhard Stengl
aus Roth bei Nürnberg



Marburg/Lahn 2006
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Vom Fachbereich Pharmazie der Philipps-Universität Marburg
als Dissertation angenommen am: 06. Juli 2006

Erstgutachter: Prof. Dr. Gerhard Klebe
Zweitgutachter: PD Dr. Klaus Reuter

Tag der mündlichen Prüfung: 06. Juli 2006 4
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Die Untersuchungen zur vorliegenden Arbeit wurden auf Anregung von Herrn Prof.
Dr. G. KLEBE am Institut für Pharmazeutische Chemie des Fachbereichs Pharmazie
der Philipps-Universität Marburg in der Zeit von Oktober 2002 bis Februar 2006
durchgeführt.











































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„Wirklich innovativ ist man nur dann,
wenn mal etwas daneben gegangen ist.“

WOODY ALLEN











für HANNA













6 Abbreviations
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Abbreviations


-10 Å Ångström ( 1Å = 10 m)
A absorption at 600 nm 600
Amp Ampicillin
aqua bidest. double destillated water
ArcTGT TGT involved in archaeosine modification
CATH Protein Structure Classification Database
(Class Architecture Topology Homology)
Cm Chloramphenicol
CMC critical micellar concentration
DMSO dimethylsulfoxid
dNTP desoxynucleosidtriphosphate
DTT dithiothreitol
E. coli Escherichia coli
Tyr ECY2 unmodified E. coli tRNA
EDTA ethylendiamintetraacetate
FAE follicle-associated epithelia
h hour
HEPES 2-[4-(2-hydroxyethyl)piperazino]ethansulfonic acid
IPTG isopropylthio-β-galactosid
kb kilo bases
kDa Dalton
K competitive inhibition constant ic
K uncompetitive constant iu
Km kanamycin
LB Luria - Bertani complex medium
-1 M molarity (mol ⋅ L )
MES 2-morpholinoethansulfonic acid
min minute
NTP nucleosidtriphosphate
PAGE polyacrylamide gel electrophoresis
PAI pathogenicity island
PEG polyethylenglycol Abbreviations 7
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PCR polymerase chain reaction
PDB PROTEIN DATA BANK
P. horikoshii Pyrococcus horikoshii
PPase inorganic pyrophosphatase
preQ 7-cyano-7-deazaguanine 0 7-aminomethyl-7-deazaguanine 1
Q 7-(((4,5-cis-dihydroxy-2-cyclopenten-1-yl)amino)
methyl)-7-deazaguanosine
QueA S-adenosylmethionine:tRNA-ribosyltransferase-
isomerase
QueTGT TGT involved in Q modification
SCOP S tructural Classification of Proteins Database
SDS sodiumdodecylsulfate
S. flexneri Shigella flexneri
SPB standard phosphate binding motif
SPR surface plasmon resonance
TCA trichloroacetic acid
TGT tRNA-guanine transglycosylase
TIM-barrel triose-phosphate isomerase (TIM) / (βα) barrel 8
T. maritima Thermotoga maritima
Tris tris-(hydroxymethyl)-aminomethan
w/v weight per volume
w.t. wild type
Asp YadB glutamyl-queuosine tRNA synthetase
Z. mobilis Zymomonas mobilis











8 Table of contents
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Table of contents

Abbreviations .......................................................................................................... 2
Table of contents .................................................................................................... 2
1. Introduction and Motivation ................................................................................. 2
1.1 Structure-based drug design and TGT........................................................... 2
1.2 Shigellosis...................................................................................................... 2
1.2.1 Disease and treatment .......................................................................... 2
1.2.2 Shigella – Escherichia relationship........................................................ 2
1.2.3 Cellular and molecular pathogenicity ................................................... 2
1.2.4 Regulation of pathogenicity................................................................... 2
1.3 Queuosine-modification 2
1.3.1 tRNA-modification ................................................................................. 2
1.3.2 Queuosine-modification pathway .......................................................... 2
1.3.3 Archaeosine-modification in Archaebacteria ......................................... 2
1.4 Aim of the project ........................................................................................... 2
2. Structural and Functional Analysis ......................................................................... 2
2.1 QueTGT – ArcTGT: base exchange reaction................................................. 2
2.1.1 TGTs in the tree kingdoms of live 2
2.1.2 Eubacterial QueTGT ............................................................................. 2
2.1.2.1 Introduction into the tRNA – QueTGT complex ................................. 2
2.1.2.2 New model for the base exchange mechanism in QueTGT .............. 2
2.1.3 Eukaryotic QueTGT .............................................................................. 2
2.1.4 Archaebacterial ArcTGT........................................................................ 2
2.1.4.1 – ArcTGT complex................................... 2
2.1.4.2 change mechanism in ArcTGT................ 2
2.2 QueTGT – ArcTGT: substrate specificity ....................................................... 2
2.2.1 QueTGT – ArcTGT: regulation of substrate specificity.......................... 2
2.2.2 QueTGT substrate selectivity – TGT(E235Q) mutant ........................... 2
2.2.2.1 Introduction........................................................................................ 2
2.2.2.2 Results .............................................................................................. 2
2.2.2.3 Discussion of the kinetic data ............................................................ 2
2.2.2.4 Discussion of TGT(E235Q) crystal structures.................................... 2
2.2.2.5 Summary and outlook........................................................................ 2
2.3 Homodimer formation in QueTGT.................................................................. 2
2.3.1 Dimer formation in solution and in crystals............................................ 2
2.3.2 Sequence comparison of 21 TGTs from different species .................... 2
2.3.3 Functional model for the QueTGT dimer............................................... 2
2.3.4 Outlook.................................................................................................. 2
2.4 Classification of the TGT superfamily............................................................. 2
2.4.1 Evolutionary origin of the TGT superfamily ........................................... 2
2.4.2 Classification within the TGT super ............................................. 2
3. Structure-based Inhibitor Design ......................................................................... 2
3.1 Modifications of the binding assay ................................................................. 2
3.1.1 Detergents effect ligand and protein solubility....................................... 2
3.1.1.1 Detergents and non-specific inhibition.......