Untersuchung der Substratspezifität der tRNA-Guanin-Transglykosylase (TGT) aus Eukaryoten und Prokaryoten und Charakterisierung der eukaryotischen TGT

Die bakterielle tRNA-Guanin-Transglycosylase (TGT) katalysiert den Austausch des genetisch kodierten Guanins in der "Wobble"-Position der tRNAsHis,Tyr,Asp,Asn gegen die prämodifizierte Base preQ1, welche dann auf der tRNA-Ebene weiter in die vollständig modifizierte Base Queuin überführt w...

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Bibliographic Details
Main Author: Biela, Inna
Contributors: Reuter, Klaus (Prof.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2014
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Bacterial tRNA-guanine transglycosylase (TGT) catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAsHis,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to the fully modified queuine base at the tRNA level. As this enzyme is essential for the pathogenicity of Shigellae, the causative agents of bacterial dysentry, it represents an attractive target for the rational design of anti-Shigellosis compounds. However, also eucaryotes and, therefore, humans possess a TGT. In contrast to the homodimeric procaryotic TGT the eucaryotic enzyme constitutes a heterodimer consisting of a catalytic and a non-catalytic subunit, with both subunits, however, being homologous to the bacterial protomer. In addition, the physiological substrate of the eucaryotic TGT is not preQ1 but the fully modified queuine, since eucaryotes are not able to synthesise queuine de novo. Rather, they acquire this base from their diet or from the gut flora. As in a mouse model eucaryotic TGT was shown to be indirectly required for the conversion of phenylalanine to tyrosine, it seems of utmost importance to create compounds inhibiting the procaryotic but not the eucaryotic enzyme. Elsewise, phenylketonurea-like secondary effects have to be expected. Obviously, a detailed knowledge about the differences between procaryotic and eucaryotic TGT is a precondition to design specific inhibitors of the bacterial enzyme. Although no crystal structure of a eucaryotic TGT has been determined yet, homology models of the Caenorhabditis elegans and the human TGT suggest that the exchange of Cys158 and Val233 in bacterial TGT (Z. mobilis TGT numbering) by valine or rather glycine in eucaryotic TGT have a large impact on substrate specificity. Accordingly, in the first part of this work, mutated variants of bacterial TGT were generated in order to investigate the influence of a Cys158Val and a Val233Cys exchange on catalytic activity and substrate specificity. The analysis of the mutated variants was carried out via determination of their enzyme kinetic parameters, via "microscale thermophoresis", via a gel shift experiment and, lastly, via crystal structure analyses. The results show that the exchange of Cys158 to valine results in a decreased affinity of the enzyme to the physiological substrate of the bacterial enzyme, preQ1. In contrast, the exchange of Val233 to glycine leads to an enlarged substrate binding pocket which is required to accommodate queuine, the physiological substrate of the eucaryotic TGT, in a conformation compatible with the intermediately covalently bound tRNA. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate. The goal of the second part of this thesis was the biochemical characterisation of the eucarytic TGT as well as the attempt to crystallise this enzyme as a basic prerequisite for crystal structure determination. For this purpose, expression plasmids were constructed allowing the recombinant production of the murine and human TGT subunits in Escherichia coli. The (separate) production and purification of the murine catalytical subunit (mQTRT1) and of the non-catalytical subunit (mQTRTD1v1) resulted in satisfying yields of ca. 4 mg per Liter of bacterial culture, each. In contrast, the purification of the recombinant human TGT subunits was significantly less successful (in each case < 0.4 mg per Liter of bacterial culture), so that the biochemical analysis as well as crystallisation trials remained largely confined to the murine enzyme. NanoESI-MS experiments confirmed the heterodimeric quarternary structure of the eucaryotic TGT and showed that, simultaneously, only one tRNA molecule can be accomodated and converted by one heterodimer. Another finding gained by nano-ESI-MS was that the catalytic QTRT1 subunit is, in the absence of its interaction partner, present as a monomer in solution. In contrast, the non-catalytic QTRTD1v1 subunit tends to form a homodimer in the absence of QTRT1. Enzyme kinetic analyses confirmed former reports of other research groups who had shown that solely the QTRT1/QTRTD1v1 heterodimer is enzymatically active, while the separate subunits are not. Thermal shift measurements finally showed that both QTRT1 and QTRTD1v1 are much more stable under high salt conditions (1000 mM NaCl) than under low salt conditions. Crystallisation trials performed with the two subunits as well as with the heterodimeric functional enzyme yielded, in the case of QTRTD1v1, reproducable crystals. When exposed to highly focussed synchrotron radiation, these crystals diffracted to ca. 2.8 Å. A data set recorded at beamline 14.1 of BESSY II (Helmholtz center, Berlin) allowed the structure determination via molecular replacement using the coordinates of Thermotoga maritima TGT (pdb code: 2ASH) as model coordinates. The refined QTRTD1v1 structure reveals that, within the crystal, the protein is present as a homodimer whose architecture strikingly resembles the homodimer of bacterial TGT. A detailed biochemical and structural knowledge of the eucaryotic TGT is a basic requirement for the design of inhibitors which specifically inhibit the bacterial TGT while leaving the eucaryotic enzyme unaffected. The recombinant production and purification of both subunits of a eucaryotic TGT as well as the determination of a first crystal structure which were achieved in the present work constitute an impotant basis for this goal.