Investigation of Specificity Determinants in Bacterial tRNA-Guanine Transglycosylase Reveals Queuine, the Substrate of Its Eucaryotic Counterpart, as Inhibitor

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.

Fragments as Novel Starting Points for tRNA‐Guanine Transglycosylase Inhibitors Found by Alternative Screening Strategies

“…This fragment, a derivative of the amino acid arginine, is amongst the most interesting fragment hits as it induces the opening of a sub‐pocket next to the preQ 1 recognition site (Figure ). This opening was previously only reported in a co‐crystallized structure of mutated variants of TGT in complex with the nucleobase queuine (PDB code 3BLO) . The latter substrate is related to preQ 1 but comprises an attached sidechain bearing an unsaturated five‐membered carbo‐cycle (Scheme ).…”

“…This opening was previously only reported in a co-crystallized structure of mutated variants of TGT in complex with the nucleobase queuine (PDB code 3BLO). [16] The latter substrate is related to preQ 1 but comprises an attached sidechain bearing an unsaturated five-membered carbo-cycle (Scheme 1). The opening of the preQ 1 sub-pocket is brought on by the shifting of the Cys158 gatekeeper residue by 4 Å from its original position in the uncomplexed enzyme ( Figure 7).…”

A single transcription factor is sufficient to induce and maintain secretory cell architecture

“…This cohort comprises genes involved in mitochondrial trafficking (Rab26, Ostm1, and Pon3) and those involved in amino acid metabolism (Aass, Bckdk, Gcdh, Qtrt1, and Wdyhv1) (Hutson 2006;Cleveland et al 2008;Wang et al 2009;Biela et al 2013;Seminotti et al 2013;Hatazawa et al 2014). The three trafficking genes seem to be involved in interactions between mitochondria and lysosomes as mentioned (Rab26 and Ostm1) and between mitochondria and the ER (Pon3).…”

In silicoidentification of metabolic enzyme drug targets inBurkholderia pseudomallei

“…Histidyl-tRNA synthetase (hisS) has been explored as a target in Trypanosoma cruzi (115). The chokepoint enzyme queuine tRNA-ribosyltransferase (tgt) incorporates the wobble base queuine into tRNA, and is also a target in Zymomonas mobilis and Shigella (116,117) Several B. pseudomallei chokepoint enzymes are involved in DNA-related processes. Two of these enzymes, encoded by the recA and dnaQ genes, are involved in the SOS pathway (118), which mediates the bacterial response to DNA damage.…”