Identification in Archaea of a Novel D-Tyr-tRNATyr Deacylase

Abstract

Tyr deacylase. Instead, it displays homology with that of a bacterial peptidyl-tRNA hydrolase. We show, however, that the archaeal PAB2349 enzyme does not act against diacetyl-Lys-tRNA Lys , a model substrate of peptidyl-tRNA hydrolase. Based on the Protein Data Bank 1YQE structure, site-directed mutagenesis experiments were undertaken to remove zinc from the PAB2349 enzyme. Several residues involved in zinc binding and supporting the activity of the deacylase were identified. Taken together, these observations suggest evolutionary links between the various hydrolases in charge of the recycling of metabolically inactive tRNAs during translation.It is usually thought that D-amino acids cannot be incorporated into proteins because of the stereospecificity of the translational machinery. The main step of D-amino acid exclusion is believed to be ensured by aminoacyl-tRNA synthetases. However, several of these enzymes, like Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae tyrosyl-tRNA synthetases, as well as E. coli tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases, have been shown capable of catalyzing the transfer of the D-isomer of their cognate amino acid onto the corresponding tRNA species in vitro (1-5). Catalytic effi- Tyr deacylase counterreacts against the toxicity associated with the formation of D-aminoacyl-tRNAs in vivo. Actually, it was shown later that, when an E. coli ⌬dtd mutant strain is exposed to the presence of 2.4 mM D-tyrosine, nearly half of the tRNA Tyr pool can be converted into metabolically inactive D-Tyr-tRNA Tyr (6). The E. coli dtd gene has homologs in many bacteria and in all of the entirely sequenced eukaryotic genomes but one, that of Encephalitozoon cuniculi (8). The dtd ortholog in S. cerevisiae (DTD1) produces a protein that catalyzes the hydrolysis of D-Tyr-tRNA Tyr in vitro. Moreover, inactivation of DTD1 enhances the sensitivity of yeast to the harmful effect of D-tyrosine (4). Thus, D-Tyr-tRNA Tyr deacylase activity appears to be widespread in the living world. Surprisingly, however, dtd/ DTD1 homologs were not detected in the available archaeal genomes (4). Therefore, either archaea do not need a D-TyrtRNA Tyr activity, or this activity is afforded by a nonorthologous enzyme. To address this question, we used Sulfolobus solfataricus to search whether it contained D-Tyr-tRNA Tyr deacylase activity. E. coli strains used in this study are listed in Table 1. In order to overexpress genes inserted in the pET15blpa plasmid, E. coli strains K37⌬recA and K37⌬TyrH⌬recA were lysogenized with the DE3 prophage (from Novagen), to obtain strains K37⌬recADE3 and K37⌬TyrH⌬recADE3, respectively. MATERIALS AND METHODS

GEK1, a gene product of Arabidopsis thaliana involved in ethanol tolerance, is a D-aminoacyl-tRNA deacylase

“…Another type of d -Tyr-tRNA Tyr deacylase (DTD2) has been discovered in archaea (7). This enzyme displays the fold of a bacterial peptidyl-tRNA hydrolase and carries two firmly bound zinc ions, crucial to the activity (7).…”

An in vitro based investigation into the cytotoxic effects of D-amino acids

“…However, it has been observed that Escherichia coli and Bacillus subtilis tyrosyl-tRNA synthetases can potentially catalyze the formation of D-tyrosyl-tRNA Tyr to the same extent as with L-enantiomer (30). Interestingly, extracts of E. coli, yeast, rabbit reticulocytes, and rat liver were shown to have an enzyme activity capable of accelerating the hydrolysis of the ester linkage of D-Tyr-tRNA to release free tRNA and D-tyrosine (31). The fate of D-aminoacyl-tRNA had never been studied previously.…”

A continuous tyrosyl-tRNA synthetase assay that regenerates the tRNA substrate

“…Unlike cyclodityrosine synthase, D-tyrosyl-tRNA deacylase is a nearly ubiquitous enzyme in bacteria, archaea, and eukaryotes [39,41,49]. Deletion of D-tyrosyl-tRNA deacylase from E. coli and S. cerevisiae results in the accumulation of D-aminoacyltRNAs, depleting the available pool of free tRNA and impairing cell growth [39,41].…”