| Queuine (Q) is a modified base that is incorporated into the anticodon of tRNAs with sequence GUN. tRNA-guanine transglycosylase (TGT) is the enzyme responsible for this incorporation of Q. Whereas eubacteria synthesize and incorporate a Q precursor into tRNA (ultimately converted to Q), eukaryotes must obtain Q as a dietary factor and incorporate it directly into tRNA. The TGT from Shigella flexneri, which causes dysentery, has been implicated as necessary for the virulence of this organism. Therefore, comparison of the human and bacterial enzymes is important in establishing TGT as a novel antibacterial target.; Unlike the eubacterial TGT, the human enzyme exists as a heterodimer (containing catalytic and regulatory subunits). Both subunits have been cloned and overexpressed; however, purification of active enzyme has not yet been achieved.; Based upon homology modeling, the active sites of the human and eubacterial TGTs are quite similar. However, four amino acids in the active site, which are quite different, have been proposed to be responsible for differential substrate recognition. To experimentally probe the role(s) of these amino acids, a series of mutants (single and multiple mutants involving C145V, T146V, P147N, and V217G) of the E. coli TGT has been constructed. Pteridines (which are inhibitors of TGT) have been used as steric probes of the active sites of these enzymes. Biopterin (a close analogue of Q) exhibits preferential inhibition of the mutant enzymes. Additionally, unlike the wild-type E. coli TGT, the V217G and C145V/T146V/V217G mutants have been found to recognize and incorporate Q into tRNA.; Examination of eubacterial and eukaryal TGT sequences revealed that the enzyme from H. pylori contains significant substitutions (relative to the eubacterial TGTs) in these four amino acids. This enzyme was subsequently cloned, overexpressed, and purified. The H. pylori TGT is inhibited by biopterin, similar to the mutant E. coli enzymes. Furthermore, this enzyme also catalyzes the incorporation of Q into substrate tRNA---the first documented evidence of a wild-type eubacterial enzyme catalyzing this reaction.; These results confirm the predictions that the active site amino acids identified by homology modeling are involved in differential substrate recognition of eukaryal versus eubacterial TGTs. |
