Publications

• Identification in archaea of a novel D-Tyr-tRNATyr deacylase.

  Ferri-Fioni M.-L., Fromant M., Bouin A.-P., Aubard C., Lazennec C., Plateau P., Blanquet S.

  Most bacteria and eukarya contain an enzyme capable of specifically hydrolyzing D-aminoacyl-tRNA. Here, the archaea Sulfolobus solfataricus is shown to also contain an enzyme activity capable of recycling misaminoacylated D-Tyr-tRNATyr. N-terminal sequencing of this enzyme identifies open reading ... >> More

  Most bacteria and eukarya contain an enzyme capable of specifically hydrolyzing D-aminoacyl-tRNA. Here, the archaea Sulfolobus solfataricus is shown to also contain an enzyme activity capable of recycling misaminoacylated D-Tyr-tRNATyr. N-terminal sequencing of this enzyme identifies open reading frame SS02234 (dtd2), the product of which does not present any sequence homology with the known D-Tyr-tRNATyr deacylases of bacteria or eukaryotes. On the other hand, homologs of dtd2 occur in archaea and plants. The Pyrococcus abyssi dtd2 ortholog (PAB2349) was isolated. It rescues the sensitivity to D-tyrosine of a mutant Escherichia coli strain lacking dtd, the gene of its endogeneous D-Tyr-tRNATyr deacylase. Moreover, in vitro, the PAB2349 product, which behaves as a monomer and carries 2 mol of zinc/mol of protein, catalyzes the cleavage of D-Tyr-tRNATyr. The three-dimensional structure of the product of the Archaeoglobus fulgidus dtd2 ortholog has been recently solved by others through a structural genomics approach (Protein Data Bank code 1YQE). This structure does not resemble that of Escherichia coli D-Tyr-tRNATyr 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-tRNALys, 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. << Less

  J. Biol. Chem. 281:27575-27585(2006) [PubMed] [EuropePMC]

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

  Wydau S., Ferri-Fioni M.-L., Blanquet S., Plateau P.

  GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the ... >> More

  GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid. Purified GEK1 protein contains two zinc ions and proves to be a broad-specific, markedly active d-aminoacyl-tRNA deacylase in vitro. Moreover, GEK1 expression is capable of functionally compensating in E. coli for the absence of endogeneous d-Tyr-tRNA(Tyr) deacylase. Possible connections between exposure of plants to ethanol/acetaldehyde and misaminoacylation of tRNA by d-amino acids are considered. << Less

  Nucleic Acids Res. 35:930-938(2007) [PubMed] [EuropePMC]

• Metabolism of D-aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae cells.

  Soutourina J., Plateau P., Blanquet S.

  In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be e ... >> More

  In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNA(Tyr) with tyrosine. Resulting d-Tyr-tRNA(Tyr) can be hydrolyzed by a d-Tyr-tRNA(Tyr) deacylase. By monitoring E. coli growth in liquid medium, we systematically searched for other d-amino acids, the toxicity of which might be exacerbated by the inactivation of the gene encoding d-Tyr-tRNA(Tyr) deacylase. In addition to the already documented case of d-tyrosine, positive responses were obtained with d-tryptophan, d-aspartate, d-serine, and d-glutamine. In agreement with this observation, production of d-Asp-tRNA(Asp) and d-Trp-tRNA(Trp) by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was established in vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNA(Tyr) deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation of d-aminoacyl-tRNA molecules and that d-Tyr-tRNA(Tyr) deacylase has a specificity broad enough to recycle any of these molecules. The same strategy of screening was applied using Saccharomyces cerevisiae, the tyrosyl-tRNA synthetase of which also produces d-Tyr-tRNA(Tyr), and which, like E. coli, possesses a d-Tyr-tRNA(Tyr) deacylase activity. In this case, inhibition of growth by the various 19 d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects of d-tyrosine and d-leucine were reinforced upon deprivation of the deacylase. This observation suggests that, in yeast, at least two d-amino acids succeed in being transferred onto tRNAs and that, like in E. coli, the resulting two d-aminoacyl-tRNAs are substrates of a same d-aminoacyl-tRNA deacylase. << Less

  J. Biol. Chem. 275:32535-32542(2000) [PubMed] [EuropePMC]

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