Computational Protein Design (CPD) is a promising method for high throughput protein and ligand mutagenesis. Recently, we developed a CPD method that used a polar-hydrogen energy function for protein interactions and a Coulomb/Accessible Surface Area (CASA) model for solvent effects. We applied this method to engineer aspartyl-adenylate (AspAMP) specificity into Asparaginyl-tRNA synthetase (AsnRS), whose substrate is asparaginyl-adenylate (AsnAMP). Here, we implement a more accurate function, with an all-atom energy for protein interactions and a residue-pairwise generalized Born model for solvent effects. As a first test, we compute aminoacid affinities for several point mutants of Aspartyl-tRNA synthetase (AspRS) and Tyrosyl-tRNA synthetase and stability changes for three helical peptides and compare with experiment. As a second test, we readdress the problem of AsnRS aminoacid engineering. We compare three design criteria, which optimize the folding free-energy, the absolute AspAMP affinity, and the relative (AspAMP-AsnAMP) affinity. The sequences and conformations are improved with respect to our previous, polar-hydrogen/CASA study: For several designed complexes, the AspAMP carboxylate forms three interactions with a conserved arginine and a designed lysine, as in the active site of the AspRS:AspAMP complex. The conformations and interactions are well maintained in molecular dynamics simulations and the sequences have an inverted specificity, favoring AspAMP over AsnAMP. The method is not fully successful, since experimental measurements with the seven most promising sequences show that they do not catalyze at a detectable level the adenylation of Asp (or Asn) with ATP. This may be due to weak AspAMP binding and/or disruption of transition-state stabilization.

Several L-aminoacyl-tRNA synthetases can transfer a D-amino acid onto their cognate tRNA(s). This harmful reaction is counteracted by the enzyme D-aminoacyl-tRNA deacylase. Two distinct deacylases were already identified in bacteria (DTD1) and in archaea (DTD2), respectively. Evidence was given that DTD1 homologs also exist in nearly all eukaryotes, whereas DTD2 homologs occur in plants. On the other hand, several bacteria, including most cyanobacteria, lack genes encoding a DTD1 homolog. Here we show that Synechocystis sp. PCC6803 produces a third type of deacylase (DTD3). Inactivation of the corresponding gene (dtd3) renders the growth of Synechocystis sp. hypersensitive to the presence of D-tyrosine. Based on the available genomes, DTD3-like proteins are predicted to occur in all cyanobacteria. Moreover, one or several dtd3-like genes can be recognized in all cellular types, arguing in favor of the nearubiquity of an enzymatic function involved in the defense of translational systems against invasion by D-amino acids.Although they are detected in various living organisms (reviewed in Ref. 1), D-amino acids are thought not to be incorporated into proteins, because of the stereospecificity of aminoacyl-tRNA synthetases and of the translational machinery, including EF-Tu and the ribosome (2). However, the discrimination between L-and D-amino acids by aminoacyl-tRNA synthetases is not equal to 100%. Significant D-aminoacylation of their cognate tRNAs by Escherichia coli tyrosyl-, tryptophanyl-, aspartyl-, lysyl-, and histidyl-tRNA synthetases has been characterized in vitro (3-9). Recently, using a bacterium, transfer of D-tyrosine onto tRNA Tyr was shown to occur in vivo (10). With such misacylation reactions, the resulting D-aminoacyl-tRNAs form a pool of metabolically inactive molecules, at best. At worst, D-aminoacylated tRNAs infiltrate the protein synthesis machinery. Although the latter harmful possibility has not yet been firmly established, several cells were shown to possess a D-tyrosyl-tRNA deacylase, or DTD, that should help them counteract the accumulation of D-aminoacyl-tRNAs. This enzyme shows a broad specificity, being able to remove various D-aminoacyl moieties from the 3Ј-end of a tRNA (4 -6, 11). Such a function makes the deacylase a member of the family of enzymes capable of editing in trans mis-aminoacylated tRNAs. This family includes several homologs of aminoacyl-tRNA synthetase editing domains (12), as well as peptidyl-tRNA hydrolase (13,14).Two distinct deacylases have already been discovered. The first one, called DTD1, is predicted to occur in most bacteria and eukaryotes (see Table 1). Inactivation of the gene of this deacylase in E. coli (dtd) or in Saccharomyces cerevisiae (DTD1) exacerbates cell growth inhibition by several D-amino acids, including D-tyrosine (6). In fact, in an E. coli ⌬dtd strain grown in the presence of 2.4 mM D-tyrosine, as much as 40% of the cellular tRNATyr pool becomes esterified with D-tyrosine (10). Homologs of dtd/DTD1 are not found in the available archae...

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

No abstract

Background: Bacterial peptidyl-tRNA hydrolase is essential in recycling of ribosome-dissociated peptidyl-tRNAs. Results: Comparing minimalist substrates and using NMR mapping, the RNA-binding site of the hydrolase is characterized. Conclusion: Interaction between the hydrolase and tRNA involves features common to all elongator tRNAs. Significance: Knowledge of a bacterial peptidyl-tRNA hydrolase⅐substrate complex may drive the search for enzyme inhibitors.