Inhibition by L-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNA^Asp and tRNA^Asn.

Abstract

Asparaginyl-tRNA formation in Pseudomonas aeruginosa PAO1 involves a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) which forms Asp-tRNA^Asp and Asp-tRNA^Asn, and a tRNA-dependent amidotransferase which transamidates the latter into Asn-tRNA^Asn. We report here that the inhibition of this ND-AspRS by L-aspartol adenylate (Asp-ol-AMP), a stable analog of the natural reaction intermediate L-aspartyl adenylate, is biphasic because the aspartylation of the two tRNA substrates of ND-AspRS, tRNA^Asp and tRNA^Asn, are inhibited with different K_i values (41 μM and 215 μM, respectively). These results reveal that the two tRNA substrates of ND-AspRS interact differently with its active site. Yeast tRNA^Asp transcripts with some identity elements replaced by those of tRNA^Asn have their aspartylation inhibited with K_i values different from that for the wild-type transcript. Therefore, aminoacyl adenylate analogs, which are competitive inhibitors of their cognate aminoacyl-tRNA synthetase, can be used to probe rapidly the role of various structural elements in positioning the tRNA acceptor end in the active site.

Keywords::

• Aspartyl-tRNA synthetase
• nondiscriminating
• tRNA^Asn
• competitive inhibitor
• aspartol-AMP
• Pseudomonas aeruginosa

Acknowledgements

We are indebted to Richard Giegé and Catherine Florentz for the plasmids expressing yeast wild-type tRNA^Asp and its C36U and C38A variants. We thank Catherine Florentz for helpful comments on this manuscript. This work was supported by grant OGP0009597 from the Natural Sciences and Engineering Research Council of Canada (NSERC) to J.L., by grant MT13564 from the Canadian Institutes of Health Research (CIHR) to P.H.R., and by grant 2003-ER-2481 from the “Fonds pour la Formation de Chercheurs et l'Aide à la Recherche du Québec” (FCAR) to R.C., P.H.R. and J.L. D.B. was a CIHR Strategic Training Program in Antibiotic Resistance doctoral fellow; P.M.A. was a doctoral fellow of the “Ministère de l'Enseignement Supérieur et de la Recherche Scientifique de Côte d'Ivoire”, and O.F. and O.C.B. were NSERC undergraduate fellows.