Abstract
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 queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering) by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. 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.
Highlights
Transfer RNA–guanine transglycosylase (Tgt, EC 2.4.2.29) catalyses the exchange of a specific guanine base in tRNA molecules by a substituted 7-deazaguanine
No crystal structure of either of these enzymes is available yet, and the only structural information concerning the active site of a eucaryotic tRNA-guanine transglycosylase (Tgt) originates from a homology model of the Caenorhabditis elegans catalytic subunit [35]
As the complete sequence of the human catalytic subunit has become available [28,36] we created a model of this orthologue using the crystal structure of Z. mobilis Tgt as a template
Summary
Transfer RNA–guanine transglycosylase (Tgt, EC 2.4.2.29) catalyses the exchange of a specific guanine base in tRNA molecules by a substituted 7-deazaguanine. Located at the ‘‘elbow’’ of the L-shaped tRNA the modification stabilises the overall tRNA structure by supporting the formation of the non-canonical Levitt base pair with cytosine 48 [2]. Both bacterial and eucaryotic Tgts are required for the introduction of the hypermodified base queuine into position 34 (the anticodon ‘‘wobble position’’) of tRNAsHis,Tyr,Asp,Asn all of which have a uracil – guanine – uracil 35 sequence in common [3,4]. No distinct function for queuine has been demonstrated yet, its presence within the tRNA anticodon suggests that it may be involved in coordinating translational fidelity and speed
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
