Caracterisation de l'aspartyl-ARNt synthetase de Pseudomonas aeruginosa: Activite, specificite et inhibition

Aminoacyl-tRNA synthetases are essential enzymes for any cell. They attach an amino acid to its cognate tRNA in a two step reaction. However, there are cases when an aminoacyl-tRNA synthetase charges two different species of tRNA. Such an enzyme is said to be non discriminating. For instance, there are non discriminating aspartol-tRNA synthetases and glutamyl-tRNA synthetases. In the organism having one or two such non discriminating enzymes, another enzyme is responsible for the conversion of the mischarged tRNAs into the correctly acylated form. This enzyme is a tRNA-dependent amidotransferase. It transfers an amide group from a donor (glutamine, ammonia or asparagine) to the incorrectly charged amino acid (aspartate or glutamate) on the tRNA, to produce a correctly charged asparaginyl-tRNAasparagine or glutaminyl-tRNAglutamine. This pathway is called the transamidation pathway. We investigated whether this pathway was present in the proteobacteria Pseudomonas aeruginosa. The transamidation pathway exists in this organism for the formation of asparaginyl-tRNAasparagine only, and not for the synthesis of glutaminyl-tRNAglutamine. This is the first known example in the gamma-proteobacteria where the amidotransferase serves only in the formation of asparaginyl-tRNA. The non discriminating aspartol-tRNA synthetase of P. aeruginosa was then characterized. Two residues were found that are individually implicated in the recognition of tRNA asparagine by the AspRS. The mutation of these residues decreased significantly the recognition of this tRNA both in vitro and in vivo. The inhibition of the aspartol-tRNA synthetase by a non-cleavable analog of the aspartyl adenylate, aspartol adenylate, was studied. We found that this inhibitor is more or less efficient depending on the species of tRNA with which the enzyme is interacting. This difference in the inhibition could be due to a difference in the mode of interaction of the enzyme with its two tRNA substrates. Our results let us speculate on the evolution of bacterial aspartol-tRNA synthetases, and on their mechanism of reaction with their substrates.