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J. Biol. Chem., Vol. 275, Issue 42, 32535-32542, October 20, 2000

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Metabolism of D-Aminoacyl-tRNAs in Escherichia coli and Saccharomyces cerevisiae Cells^*

Julie Soutourina, Pierre Plateau, and Sylvain Blanquet

From the Laboratoire de Biochimie, Unité Mixte de Recherche 7654, CNRS-Ecole Polytechnique, 91128 Palaiseau Cedex, France

Received for publication, June 14, 2000, and in revised form, July 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

D-Amino acids are widely distributed in living organisms (1). Examples of D-amino acid natural occurrence include D-Ala- and D-Glu-containing peptidoglycans of bacterial cell walls, D-amino acid-containing natural peptide antibiotics (1-5), or the presence of D-Asp and D-Ser at high concentrations in human brain (6-10). D-Ala results from the transformation of L-Ala by a racemase. In the case of D-Glu production, a racemase or a transaminase is involved, depending on the bacterium. Small amounts of D-amino acids can also possibly appear as side products of various biosynthetic pathways. Conversion of the L- to the D-stereoisomer of tryptophan was observed in the presence of tryptophan synthase (11, 12). Similarly, in the case of methionyl-tRNA synthetase, a weak catalysis of -carbon hydrogen-deuterium exchange of L-methionine was evidenced in vitro (13). Such an exchange suggests possible conversion of L-methionine into D-methionine at the surface of an amino acid-binding enzyme. As discussed earlier (14), D-tyrosine might arise at the step of the addition of an amino group to 4-hydroxyphenylpyruvate. Moreover, D-amino acids are likely to be nonspecifically formed as side reaction products in the presence of pyridoxal phosphate-containing enzymes or of pyridoxal phosphate alone (15, 16).

If externally added, D-amino acids exert toxicity toward many organisms (1, 17-26). Possible causes of this toxicity are multiple. For instance, in the case of Escherichia coli, D-amino acids can be lethal because they are erroneously incorporated in peptidoglycan (23, 25, 26). In the case of Bacillus subtilis, strains capable of efficiently pumping D-tyrosine have been described. The growth of such strains is decreased upon addition of this D-amino acid to the culture medium (19). Inhibition of prephenate dehydrogenase and the consequent curtailment of L-tyrosine biosynthesis may account for this behavior (18). However, incorporation of D-tyrosine into proteins could be evidenced with the above B. subtilis strains (18).

In agreement with this observation, several studies indicate D-tyrosyl-tRNA^Tyr formation in E. coli (27-29) as well as in Saccharomyces cerevisiae (14). Moreover, to recycle tRNA^Tyr esterified by D-tyrosine and/or to minimize D-tyrosine incorporation into proteins, E. coli and S. cerevisiae harbor a gene (dtd or DTD1, respectively) encoding a deacylase capable of hydrolyzing D-Tyr-tRNA^Tyr into free tRNA^Tyr and D-tyrosine. In vitro, the E. coli deacylase also hydrolyzes D-Phe-tRNA^Phe and, to a lesser extent, Gly-tRNA^Gly (28). Upon inactivation of these genes, cell growth becomes sensitive to the presence of D-tyrosine in the culture medium (14, 29).

In the present study, the possibility that D-Tyr-tRNA^Tyr deacylase could be involved in the deacylation of other D-aminoacyl-tRNAs distinct from D-Tyr-tRNA^Tyr was investigated in vivo. In a first experiment, toxicities of each D-amino acid on the growth of E. coli dtd⁺ and dtd strains were systematically compared. An involvement of the dtd product in the protection of E. coli against the toxicity of D-Trp, D-Asp, D-Ser, and D-Gln was revealed. In the case of D-Trp and D-Asp, in vitro studies established that D-Trp-tRNA^Trp and D-Asp-tRNA^Asp could be produced by the corresponding aminoacyl-tRNA synthetases. Moreover, the two D-aminoacyl-tRNAs were efficient substrates of purified D-Tyr-tRNA^Tyr deacylase.

In the case of S. cerevisiae, comparison of the growth of DTD1 and dtd1 strains indicated protection against D-leucine afforded by the D-Tyr-tRNA^Tyr deacylase gene.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

L-[5-³H]Tryptophan (740 GBq/mmol), L-[2,3-³H]aspartic acid (703 GBq/mmol), D-[2,3-³H]aspartic acid (666 GBq/mmol), and [³²P]-PP_i (300 GBq/mmol) were from PerkinElmer Life Sciences. Non-radioactive D- and L-amino acids were from Sigma. Q-Sepharose and Superose 6 were from Amersham Pharmacia Biotech. Hydroxylapatite was from Bio-Rad. 214TP5215 C₄ column (2.1 × 150 mm, 5-µm particles) was from Vydac. Bakerbond Wide-Pore Butyl (C₄) (15 µm particles) was from Baker. Crude E. coli tRNA was from Roche Molecular Biochemicals.

Purification of E. coli Tryptophanyl-tRNA Synthetase-- To overproduce tryptophanyl-tRNA synthetase, the trpS gene of plasmid pCH17 (Table I) was inserted into pBluescript under the control of the lacZ promoter. For this purpose, the 3.2-kilobase pair SalI-EcoRI fragment of pCH17 was subcloned between the XhoI and EcoRI sites of pBluescript()KS. Cells transformed by the resulting plasmid (pBStrpS) were used as a source of enzyme.

Cells were grown overnight at 37 °C in 4 liters of 2× TY (30) medium containing 200 µg of ampicillin/ml. Crude extract preparation, nucleic acid precipitation with streptomycin sulfate, and ammonium sulfate precipitation of proteins were performed as described previously (29). After the ammonium sulfate precipitation, the protein pellet was dissolved in 20 ml of 20 mM potassium phosphate (pH 6.75), containing 0.1 mM EDTA and 10 mM 2-mercaptoethanol (buffer A), and dialyzed against 2 liters of the same buffer. The resulting sample was applied on a Q-Sepharose column (2.6 × 16 cm), which was eluted at a flow rate of 180 ml/h with a 1.35-liter linear gradient from 0 to 300 mM KCl in buffer A. Tryptophanyl-tRNA synthetase was recovered at 120 mM KCl. Fractions showing tryptophan-dependent ATP-PP_i exchange activity were directly applied on a hydroxylapatite column (2.6 × 10 cm) equilibrated in buffer A. This column was eluted at a flow rate of 20 ml/h using a 2-liter linear gradient from 20 to 500 mM potassium phosphate (pH 6.75). Tryptophanyl-tRNA synthetase activity was recovered at 220 mM potassium phosphate and concentrated by an ammonium sulfate precipitation (70% saturation). After centrifugation at 10,000 × g for 30 min, the pellet was dialyzed against 20 mM Tris-HCl (pH 7.8) containing 60% glycerol, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol, and stored at 20 °C. 230 mg of enzyme were recovered from 15.2 g of cells (wet weight), with a yield of 70%.

According to SDS-polyacrylamide gel electrophoresis analysis, purified tryptophanyl-tRNA synthetase was at least 95% homogeneous. Concentration of tryptophanyl-tRNA synthetase was calculated using a M_r of 2 × 37,497 and a light absorption coefficient of 0.739 A₂₈₀ units mg¹ ml, as deduced from the amino acid sequence of the protein.

Purification of E. coli Aspartyl-tRNA Synthetase-- Aspartyl-tRNA synthetase was purified from E. coli strain JM101TR transformed by plasmid pBluescript()KSaspS (pBSaspS) (31). Cells were grown overnight at 37 °C, in 1 liter of 2× TY medium containing 500 µg of ampicillin/ml. Crude extract preparation and streptomycin sulfate and ammonium sulfate precipitations followed the protocol described previously (29). After the ammonium sulfate precipitation, the protein pellet was dissolved in 5 ml of 20 mM Tris-HCl (pH 7.8), containing 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and 50 mM KCl (buffer B), and dialyzed against 2 liters of the same buffer for 2 h. Then, the protein sample was applied on a Superose 6 column (2 × 50 cm) equilibrated in buffer B. The column was eluted at a flow rate of 12 ml/h with 112 ml of buffer B. Fractions showing aspartyl-tRNA synthetase tRNA aminoacylation activity were pooled and directly applied on a Q-Sepharose Hi-Load column (1.6 × 10 cm) equilibrated in buffer B. The column was eluted at a flow rate of 150 ml/h with a 260-ml linear gradient from 50 to 400 mM KCl in buffer B. Enzyme activity was recovered at 275 mM KCl. After ammonium sulfate precipitation (70% saturation) and centrifugation at 10,000 × g for 30 min, the pellet was dialyzed against 20 mM Tris-HCl (pH 7.8) containing 60% glycerol, 0.1 mM EDTA, and 10 mM 2-mercaptoethanol, and stored at 20 °C. 30 mg of enzyme were recovered from 2.6 g of cells (wet weight) with a yield of 45%.

Recovered aspartyl-tRNA synthetase was at least 95% homogeneous, according to SDS-polyacrylamide gel electrophoresis analysis. Enzyme concentration was determined using a M_r of 2 × 65,913 and a light-absorption coefficient of 0.589 A₂₈₀ units mg¹ ml, as calculated from the amino acid sequence of the protein.

Purification of E. coli tRNA^Trp-- The E. coli tRNA^Trp gene (trpT) was constructed and overexpressed in strain JM101TR (Table I) from a pBSTNAV derivative, according to a previously described method (32).

Since E. coli tRNA^Trp renaturation requires exposure to heat in the presence of Mg²⁺ (33, 34), the crude tRNA extract from the above cells was dissolved in 20 mM Tris-HCl (pH 7.8) containing 10 mM MgCl₂ and 0.1 mM EDTA, and heated for 5 min at 60 °C. Upon this treatment, the L-tryptophan acceptance of the tRNA sample increased from 370 to 600 pmol of L-tryptophan/A₂₆₀ unit.

The tRNA^Trp sample was further purified on a preparative C₄ column. A set of 10 samples of 70 A₂₆₀ units each were successively chromatographed on a 20-ml Bakerbond C₄ column (1.6 × 10 cm) equilibrated in 1 M sodium formate buffer (pH 5.5) containing 10 mM NaH₂PO₄ and 8 mM MgCl₂ (solution C). The column was eluted at a flow rate of 0.5 ml/min with (i) a 15-ml linear gradient from 0% to 20% of a solution containing 10 mM NaH₂PO₄ (pH 5.5) plus 10% methanol (solution D), (ii) a 20-ml linear gradient from 20% to 60% of solution D, and (iii) a 10 ml linear gradient from 60% to 100% of solution D. Three peaks of L-tryptophan acceptance activity were recovered at 640, 440, and 140 mM sodium formate concentrations (36%, 56%, and 86% of solution D, respectively). With a crude tRNA extract from a non-overproducing strain, one single peak was observed at 140 mM sodium formate. Therefore, the first two peaks are likely to reflect undermodified stages of the overproduced tRNA^Trp and were discarded. After dialysis against 3 liters of a 20 mM Tris-HCl buffer (pH 7.8) containing 1 mM MgCl₂ and 0.1 mM EDTA, the tRNA sample corresponding to 140 mM sodium formate was precipitated with ethanol and stored. Before use, the tRNA^Trp pellet was recovered by centrifugation, dissolved in the above buffer, and heated for 5 min at 60 °C. Purified tRNA^Trp accepted 1400 pmol of L-tryptophan/A₂₆₀ unit.

Purification of E. coli tRNA^Asp-- The E. coli tRNA^Asp gene (aspT) was constructed and overexpressed in JM101TR cells as described above in the case of tRNA^Trp. The resulting crude tRNA extract accepted 700 pmol of L-aspartic acid/A₂₆₀ unit.

The tRNA^Asp sample was further purified on a 20-ml Bakerbond C₄ column, as described above for tRNA^Trp. One single peak of L-Asp acceptance activity was recovered in agreement with the already described full post-transcriptional modification of overproduced tRNA^Asp (35). After C₄ chromatography, the tRNA sample was precipitated with ethanol and stored. Purified tRNA^Asp accepted 1000 pmol of L-aspartic acid/A₂₆₀ unit.

Aminoacyl-tRNA Synthetase Activity Assays-- Tryptophan-dependent ATP-PP_i exchange activity was assayed during 10 min at 28 °C in 100 µl of a reaction mixture containing 20 mM Tris-HCl (pH 7.8), 7 mM MgCl₂, 2 mM ATP, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 2.5 mM 2-mercaptoethanol, 2 mM L-tryptophan, 2 mM [³²P]PP_i (20-80 MBq/mmol), and catalytic amounts of enzyme. After quenching of the reaction, labeled ATP was adsorbed on charcoal, filtered, and counted as described previously (36).

Aminoacylation of tRNA^Trp with L- or D-tryptophan was assayed during 10 min at 28 °C in 100 µl of a reaction mixture containing 20 mM Tris-HCl (pH 7.8), 7 mM MgCl₂, 2 mM ATP, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 2.5 mM 2-mercaptoethanol, 1.8-3.6 µM L- or D-tryptophan, 0.35 µM purified tRNA^Trp, and catalytic amounts of tryptophanyl-tRNA synthetase. The reaction was quenched by the addition of 10 µl of 3 M sodium acetate (pH 5.5) plus 55 µl of phenol saturated with a 20 mM sodium acetate solution (pH 5.3). After vigorous shaking and centrifugation, the aqueous phase was precipitated with ethanol. The samples were chromatographed on a C₄ HPLC¹ column as described below. Light absorption of the elution profile was measured at 260 nm. Concentrations of aminoacylated and non-aminoacylated tRNA^Trp were deduced from the areas of the corresponding absorption peaks.

Aminoacylation of tRNA^Trp with L-[³H]tryptophan (2500 Ci/mol) was also measured by scintillation counting. In this case, the reaction was quenched by the successive addition of 2.5 ml of ice-cold trichloroacetic acid (5%, w/w) containing 0.5% tryptophan, and of 10 µl of carrier RNA from yeast (4 mg/ml). The precipitate was recovered on a Whatman GF-C filter, and the retained radioactivity was measured in a Beckman LS1801 scintillation counter.

Aminoacylation of tRNA^Asp by aspartyl-tRNA synthetase was assayed by scintillation counting, as described above for tryptophanyl-tRNA synthetase, except that 5 µM tRNA^Asp and 1 µM either L- or D-[³H]aspartic acid (500 Ci/mol) were used in the assay. Ice-cold trichloroacetic acid without amino acid was added to quench the reaction.

HPLC of Aminoacylated tRNA^Trp-- The tRNA samples (0.025-2 A₂₆₀ units) were dissolved in 100 µl of a 3 M sodium formate buffer (pH 5.5) containing 10 mM NaH₂PO₄ and 8 mM MgCl₂. Then, each sample was loaded on C₄ HPLC column (2.1 × 150 mm) equilibrated with solution C. To recover the tRNA and its aminoacylated derivative, the eluant of the column was progressively changed to solution D, through a 6.75-ml linear gradient from 0% to 30% of solution D, followed by a 12-ml linear gradient from 30% to 100% of solution D. The absorbance of the column effluent was measured at 260 nm.

Preparation of Aminoacylated tRNAs-- For D-Trp-tRNA^Trp preparation, a reaction mixture (4 ml) containing 20 mM Tris-HCl (pH 7.8), 7 mM MgCl₂, 2 mM ATP, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 2.5 mM 2-mercaptoethanol, 0.35 µM of purified tRNA^Trp, 3.6 µM D-tryptophan, and 2 µM tryptophanyl-tRNA synthetase was incubated at 28 °C for 10 min. After phenol extraction and ethanol precipitation, the sample was purified by C₄ HPLC, as described above. Fractions containing D-Trp-tRNA^Trp were pooled and precipitated with ethanol. L-[³H]Trp-tRNA^Trp was prepared under the same conditions, except that the reaction mixture (1.2 ml) contained 0.35 µM purified tRNA^Trp (0.3 A₂₆₀ units), 10 µM L-[³H]tryptophan (100 Ci/mol), and 1 µM tryptophanyl-tRNA synthetase.

For L-[³H]Asp-tRNA^Asp preparation, a reaction mixture (300 µl) containing 20 mM Tris-HCl (pH 7.8), 7 mM MgCl₂, 2 mM ATP, 0.1 mM EDTA, 50 µg/ml of bovine serum albumin, 2.5 mM 2-mercaptoethanol, 1 µM purified tRNA^Asp, 30 µM L-[³H]aspartic acid (500 Ci/mol), and 1.5 µM aspartyl-tRNA synthetase was incubated at 28 °C for 10 min. After phenol extraction and ethanol precipitation, the product was purified by chromatography on a Trisacryl GF05 column, as described previously for Tyr-tRNA^Tyr (29).

Because the D-aspartic acid sample was contaminated by L-aspartic acid (see "Results"), the preparation of D-aspartyl-tRNA was achieved in two steps. First, the reaction mixture (1 ml) containing 20 mM Tris-HCl (pH 7.8), 7 mM MgCl₂, 2 mM ATP, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 2.5 mM 2-mercaptoethanol, 60 µM D-[³H]aspartic acid (500 Ci/mol), 5 µM purified tRNA^Asp, and 0.325 µM aspartyl-tRNA synthetase was incubated at 28 °C for 10 min. Under these conditions, most of the contaminating L-aspartic acid was exhausted through rapid transfer onto the tRNA and practically no D-aspartic acid was consumed for aminoacylation. After precipitation with ethanol, the supernatant containing L-Asp-free D-[³H]aspartic acid was lyophilized, suspended in water, and further used in an aminoacylation reaction at 28 °C for 20 min with 5 µM purified tRNA^Asp and 6.5 µM aspartyl-tRNA synthetase (second step). After phenol extraction and ethanol precipitation, the sample was purified on a Trisacryl GF05 column, as described (29).

D-Tyr-tRNA^Tyr Deacylase Activity Measurements-- Initial rates of D- or L-Trp-tRNA^Trp deacylation were measured for 5 min at 28 °C in 100-µl assays containing 0.1-0.3 µM D-Trp-tRNA^Trp or L-[³H]Trp-tRNA^Trp, 20 mM Tris-HCl (pH 7.8), 5 mM MgCl₂, 0.1 mM EDTA, and catalytic amounts of purified E. coli D-Tyr-tRNA^Tyr deacylase (29). The reaction was quenched by addition of phenol. Then, the aqueous phase was precipitated with ethanol and analyzed by C₄ HPLC. Initial rates of L-[³H]Trp-tRNA^Trp hydrolysis were also measured by scintillation counting, as described previously for tyrosyl-tRNA hydrolysis (29).

Initial rates of D- or L-[³H]-Asp-tRNA^Asp hydrolysis by the deacylase were measured by scintillation counting (29).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Toxicity of D-Tryptophan Depends on the Presence or Absence of D-Tyr-tRNA^Tyr Deacylase-- Preliminary experiments suggested that the growth of a dtd strain exhibited enhanced sensitivity to the presence of D-tryptophan, when compared with an isogenic dtd⁺ strain (29). This behavior was confirmed by plating strains K37TyrH (dtd::kan, Table I) and K37 (dtd⁺) on M9 minimal medium agar plates (30) supplemented with 0.2% glucose and 5 mM D-tryptophan. In these conditions, the growth of the dtd strain was almost completely abolished, while the dtd⁺ strain still grew (Fig. 1).

View larger version (151K): [in this window] [in a new window]   Fig. 1.   Effect of D-tryptophan on the growth of E. coli strains K37 (dtd+) and K37TyrH (dtd). Cells were left to grow for 40 h at 37 °C on M9 minimal medium agar plates supplemented with the indicated concentrations of D-tryptophan (0, 0.2, 0.8, and 5 mM).

Therefore, it could be suspected that D-tryptophan, similarly to D-tyrosine (29), was transferred onto its specific tRNA in vivo, and that the resulting D-Trp-tRNA^Trp was deacylated by D-tyrosyl-tRNA deacylase.

Toxicity Associated with Several Other D-Amino Acids Also Depends on the Presence of the Deacylase-- To determine whether the D-tyrosyl-tRNA deacylase protected E. coli cells against D-amino acids other than D-tyrosine or D-tryptophan, we studied the effect of 18 D-amino acids on the growth of strains K37 (dtd⁺) and K37TyrH (dtd::kan) (Table II). Because of its limited solubility, D-tyrosine could not be included in this toxicity test. Bacteria were inoculated at a final OD₆₅₀ of 0.003 in liquid M9 minimal medium supplemented with 0.2% glucose and various concentrations of the D-amino acid under study (0-25 mM). After an 8-h incubation at 37 °C, the optical density of the culture was compared with that of a control culture without added D-amino acid.

View this table: [in this window] [in a new window]   Table II Effect of various D-amino acids on the growth of E. coli strains K37 and K37TyrH Cells were pre-grown to mid-exponential phase (0.2-0.3 OD650) in M9 minimal medium without D-amino acid. Then, they were inoculated at an optical density of 0.003 at 650 nm in liquid M9 minimal medium containing or not different D-amino acid concentrations. After an 8-h incubation at 37 °C, the optical densities of the cultures were measured at 650 nm. In the control culture without D-amino acid, the optical density amounted to 0.3-0.4. D-amino acid concentrations were varied from 1 µM to 512 µM in the case of D-Val, D-Cys, D-Trp, and from 0.05 mM to 25 mM with the other D-amino acids. Successive concentrations differed by a factor of 2. Because of its low solubility, D-tyrosine was omitted from this test. Indicated in the table are the D-amino acid concentrations causing a 2-fold decrease in the optical density at the end of incubation, as compared to the control culture without D-amino acid.

Under these conditions, no inhibition of the bacterial growth was observed upon addition of D-Ala, D-Pro, or D-Arg. With D-Asn, D-His, D-Ile, D-Leu, D-Met, and D-Phe, the toxic effect was small and no difference could be noted between strains K37 and K37TyrH. Several D-amino acids (D-Glu, D-Thr, D-Lys, D-Val, and D-Cys) markedly inhibited E. coli growth (Table II). However, the toxicity of these amino acids was the same with the two strains.

Finally, a difference in the behaviors of dtd and dtd⁺ strains was observed in the presence of D-Asp, D-Ser, D-Gln, or, as expected, of D-Trp (Table II). The toxicity of these D-amino acids was significantly more pronounced when the dtd gene was lacking. Consequently, we suspected that these D-amino acids are transferred onto their specific tRNA in vivo and that the resulting aminoacyl-tRNAs are deacylated by D-Tyr-tRNA^Tyr deacylase. These two hypotheses were verified in the case of D-tryptophan, a hydrophobic amino acid larger than D-tyrosine, and in the case of the small ionizable D-aspartic acid.

E. coli Tryptophanyl-tRNA Synthetase Catalyzes Aminoacylation of tRNA^Trp with D-Tryptophan-- Because radioactive D-tryptophan was not commercially available, we monitored aminoacylation of tRNA^Trp by D-tryptophan with the help of HPLC. Enzymatic assays were performed with purified tRNA^Trp and purified tryptophanyl-tRNA synthetase. The chromatogram of a control reaction without added synthetase is shown in Fig. 2A. Assignation of the peak at 52 min to tRNA^Trp was obtained by collecting fractions and assaying them for L-tryptophan acceptance. Upon incubation of the tRNA sample in the presence of 1.8 µM L-[³H]tryptophan and 1 nM tryptophanyl-tRNA synthetase prior to the chromatography, an additional peak became visible at 68 min on the chromatogram (Fig. 2B). This peak was assigned to Trp-tRNA^Trp because (i) the fractions corresponding to this peak contained radioactivity and (ii) the quantity of L-Trp-tRNA as calculated from the peak area corresponded to the quantity measured by trichloroacetic acid precipitation and counting before application on the column.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   C4-HPLC analysis of tRNATrp aminoacylation (A-C) and of D-Trp-tRNATrp hydrolysis (D-F). The absorbance of the column effluent was measured at 260 nm. A, tRNATrp (0.35 µM) incubated without tryptophanyl-tRNA synthetase and without tryptophan. B, tRNATrp incubated for 10 min in the presence of 1.8 µM L-[3H]tryptophan and 1 nM synthetase. C, tRNATrp incubated for 10 min in the presence of 1.8 µM D-tryptophan and 300 nM synthetase. The minor peak at 46 min corresponds to non-aminoacylatable material in the sample. D, D-Trp-tRNATrp (0.3 µM) before incubation. E, D-Trp-tRNATrp incubated for 5 min in the presence of 200 pM D-tyrosyl-tRNATyr deacylase. F, D-Trp-tRNATrp incubated for 5 min in the presence of 30 nM deacylase.

When D-tryptophan was used as substrate, a peak migrating at 68 min was also obtained on the chromatogram (Fig. 2C). The area of this peak was used to calculate the concentration of the D-Trp-tRNA^Trp product. Initial rate of D-Trp-tRNA^Trp formation in the presence of 1.8 µM D-tryptophan and 0.35 µM tRNA^Trp was 1.1 × 10³ s¹. In the presence of excess enzyme, maximum esterification of D-tryptophan to tRNA reached 0.18 µM. Since the optical purity of the D-amino acid sample is >98%, this value guaranteed that more than 80% of the esterified amino acid corresponded to the D-stereoisomer. Under the same assay conditions, initial rate of tRNA aminoacylation with L-tryptophan was 0.16 s¹ (Table III).

D-Trp-tRNA^Trp Is Hydrolyzed by D-Tyr-tRNA^Tyr Deacylase-- As explained above, the enhanced sensitivity of a dtd strain to the presence of D-tryptophan suggested that D-Tyr-tRNA^Tyr deacylase can hydrolyze D-Trp-tRNA^Trp. To explore this possibility, D-Trp-tRNA^Trp was purified by C₄ HPLC and assayed as a substrate of the deacylase. After incubation in the presence of the enzyme, aminoacylated and non-aminoacylated tRNAs were separated by HPLC and quantitated. Typical chromatograms are shown in Fig. 2 (D-F). Incubation for 5 min in the presence of a 200 pM enzyme concentration resulted in the hydrolysis of 28% of the initially added D-Trp-tRNA^Trp (0.3 µM) (Fig. 2E). At 30 nM enzyme (Fig. 2F), deacylation was practically complete (91%) in 5 min. When the incubation was carried out without enzyme, only 10% of the substrate were deacylated. From such experiments, it could be concluded that D-Tyr-tRNA^Tyr deacylase promotes the hydrolysis of D-Trp-tRNA^Trp, with a catalytic efficiency (k_cat/K_m) equal to 2.8 µM¹ s¹ (Table III). Calculated rate of spontaneous chemical hydrolysis was 3 × 10⁴ s¹.

The deacylase did not hydrolyze L-Trp-tRNA^Trp at a significant rate (k_cat/K_m<10³ µM¹ s¹) (Table III). The different behaviors of the deacylase in the presence of D- or L-Trp-tRNA^Trp further show that the tRNA aminoacylation reaction obtained with D-tryptophan did not result from an L-tryptophan contamination in the D-amino acid sample.

E. coli Aspartyl-tRNA Synthetase Catalyzes the Aminoacylation of tRNA^Asp with D-Aspartic Acid-- The formation of D-Asp-tRNA^Asp was investigated in the presence of 1 µM [³H]-radiolabeled D-aspartic acid (500 Ci/mol), 5 µM purified tRNA^Asp, and purified aspartyl-tRNA synthetase. In these conditions, the kinetics of incorporation of radioactivity into tRNA were biphasic. First, 1.4% of total ³H in the sample was rapidly transferred onto tRNA. Then, incorporation of ³H continued at a much slower rate. The first part of the kinetic was likely to correspond to the rapid esterification of tRNA^Asp by L-aspartic acid contaminating the D-aspartic acid. The second phase was attributed to the formation of D-Asp-tRNA^Asp. A 1.4% contamination was compatible with the given optical purity of D-[³H]aspartic acid (>97%). In agreement with this hypothesis, only the first phase of the kinetics was still observed if 150 nM E. coli D-Tyr-tRNA^Tyr deacylase was added to the tRNA aminoacylation assay mixture. Indeed, since the deacylase is expected to hydrolyze specifically D-Asp-tRNA^Asp, its addition in the aminoacylation assay prevents the accumulation of the D-Asp-tRNA^Asp product.

From the above experiments, we measured an initial rate of esterification of tRNA^Asp (5 µM) by D-Asp (1 µM) equal to 0.8 × 10⁵ s¹. Under the same experimental conditions, L-[³H]aspartic acid was transferred onto tRNA^Asp at an initial rate of 3.3 × 10² s¹ (Table III).

D-Asp-tRNA^Asp Is Hydrolyzed by D-Tyr-tRNA^Tyr Deacylase-- To measure the initial rate of D-Asp-tRNA hydrolysis by the deacylase, D-[³H]Asp-tRNA was prepared and assayed as a substrate of this enzyme. As explained under "Materials and Methods," care was taken to minimize contamination of D-[³H]Asp-tRNA^Asp by L-[³H]Asp-tRNA^Asp.

A value of 12 µM¹ s¹ was obtained for the catalytic efficiency (k_cat/K_m) of D-Asp-tRNA^Asp hydrolysis by the deacylase (Table III). This value is 4-fold greater than the catalytic efficiency measured with the D-Trp-tRNA^Trp substrate (2.8 µM¹ s¹), and 2-fold higher than that obtained in the presence of D-Tyr-tRNA^Tyr (6 µM¹ s¹) (29). L-Asp-tRNA^Asp was not a substrate of D-Tyr-tRNA^Tyr deacylase (k_cat/K_m<10³ µM¹ s¹). In the incubation mixture, spontaneous chemical hydrolysis of either D- or L-Asp-tRNA^Asp occurred at a same rate equal to 2 × 10⁴ s¹.

Effect of D-Amino Acids on the Growth of S. cerevisiae DTD1 or dtd1 Strains-- In S. cerevisiae, the DTD1 gene product is structurally and functionally homologous to E. coli D-Tyr-tRNA^Tyr deacylase (14). Disruption of the DTD1 gene markedly increases the sensitivity of the growth of yeast to the presence of external D-tyrosine.

To assess the specificity of the yeast D-Tyr-tRNA^Tyr deacylase, the effects of 19 D-amino acids on the growth of the S. cerevisiae DTD1 (DBY2057) and dtd1 (DBY2057DTD1) strains were compared (Table IV). Because limiting nitrogen conditions enhance the sensitivity of yeast growth to various D-amino acids (37-39), L-proline was used as nitrogen source in our experiments. Yeast cells were left to grow at 30 °C on minimal medium agar plates (yeast nitrogen base without amino acids and ammonium sulfate, from Difco) supplemented with uracil, glucose, L-proline, and various concentrations of D-amino acids (0-10 mM).

View this table: [in this window] [in a new window]   Table IV Effect of various D-amino acids on the growth of S. cerevisiae strains DBY2057 and DBY2057DTD1 Cells were grown at 30 °C on minimal medium agar plates supplemented with uracil, glucose, L-proline, and D-amino acids at different concentrations. Explored D-amino acid concentrations were 0.01, 0.03, 0.1, 0.3, 1, 3, 5, and 10 mM. Because of its low solubility, D-tyrosine could not be tested beyond 0.3 mM. Indicated in the table are the minimal D-amino acid concentrations resulting in the complete absence of growth after a 6-day incubation.

The addition of up to 10 mM D-Pro, D-Asn, or D-Ile to the minimal medium did not modify the growth of the cells. Little growth inhibition was observed with D-Cys, D-Thr, D-Glu, D-Asp, D-Val, D-Lys, and D-Gln. The inhibition was the same with wild type or dtd1 cells (Table IV).

Cell growth was clearly sensitive to the presence of D-Ser, D-Ala, D-Arg, D-Trp, D-Met, D-Phe, and D-His, in order of increasing toxicity. However, again, the DTD1 and dtd1 strains were affected to similar extents.

Finally, upon addition of D-leucine to the growth medium, a marked difference could be observed between the two strains. Although growth of the wild-type strain was insensitive to the presence of up to 10 mM D-leucine, that of the dtd1 strain was stopped by the addition of 0.3 mM D-leucine.

As a control, the DTD1 and dtd1 strains were also exposed to D-tyrosine. As shown in Table IV, full inhibition of the growth of the dtd1 strain was reached at 0.1 mM D-tyrosine. With the DTD1 strain, identical growth inhibition required 0.3 mM D-amino acid.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This study enables us to draw two main conclusions. First, aminoacyl-tRNA synthetases are less specific than generally believed, since D-amino acids succeed in being transferred onto tRNAs. Second, the specificity of the previously characterized D-tyrosyl-tRNA^Tyr deacylase is much broader than expected.

By analogy with the case of D-tyrosine, the screening for D-amino acid toxicity, as shown here, suggests aminoacylation by their cognate D-amino acid of tRNA^Trp, tRNA^Asp, tRNA^Ser, and tRNA^Gln in E. coli, and of tRNA^Leu in yeast. In the cases of tRNA^Trp and tRNA^Asp, the transfer reaction could be demonstrated in vitro. However, the measured rates of D-aminoacylation are slow. They are smaller than the rates of L-aminoacylation by 1-3 orders of magnitude, depending on the considered aminoacyl-tRNA synthetase. Such low rates are likely to reflect relatively high K_m and small k_cat values associated with D-amino acids in the formation of D-aminoacyl-adenylates, as well as possible impairments at the level of the transfer reaction itself. On the other hand, the D-amino acids are not likely to affect tRNA binding.

D-Tryptophan and D-Phenylalanine-- The observed toxic effects of D-amino acids on E. coli growth may result from several mechanisms in addition to a mischarging of tRNA. For instance, the toxicity of D-tryptophan toward E. coli growth is acute. Possibly, part of this toxicity is contributed for by misincorporation of D-tryptophan in the bacterium cell walls (23, 25, 26). The mechanism of this incorporation does not imply tRNA^Trp esterification. It is independent of the normal biosynthetic pathway and apparently involves a penicillin-insensitive LD-transpeptidase enzyme (25, 26). The harmful effect of D-tryptophan incorporation into cell walls is explained by an increased susceptibility of modified peptidoglycan to the action of lytic transglycosylases, the main autolytic enzymes in E. coli (25). D-Tryptophan can also inhibit the formation of lipoprotein-peptidoglycan linkage (23).

Similarly to D-tryptophan, D-phenylalanine exerts toxicity through misincorporation in cell walls (25, 26). In our experiments, the effect of D-phenylalanine does not depend on the presence or absence of the dtd allele. However, this D-amino acid was shown early to be esterified to tRNA by phenylalanyl-tRNA synthetase in vitro (28). Hence, we conclude that, even if Phe-tRNA^Phe was produced in vivo, the inhibitory effect of this molecule on E. coli growth is negligible as compared with the perturbation of cell wall synthesis induced by the D-amino acid.

D-Serine Toxicity-- The present study indicates that the toxicity of D-serine is at least partially contributed for by the formation of a D-Ser-tRNA. Nevertheless, D-serine may also behave as a powerful feedback inhibitor of L-serine and pantothenate biosyntheses (40). It is noteworthy that the bacterium is able to counter-react against the in vivo accumulation of D-serine through the action of D-serine deaminase. Moreover, in the presence of D-serine or of its analog D-threonine, D-serine deaminase expression is induced via a specific activator protein (dsdC gene product) and the catabolite gene activator protein-cAMP complex. Under conditions of full induction, the D-serine deaminase activity becomes high enough to allow growth in the presence of D-serine as the only nitrogen and carbon source (40, 41). The efficiency of this enzyme may explain why addition of millimolar concentrations of D-serine are required to evidence toxicity against E. coli.

Other D-Amino Acids-- The screening also shows an effect of relatively small concentrations of D-Glu, D-Thr, D-Lys, D-Val, and D-Cys on the growth of E. coli. The toxicity of the above five D-amino acids may originate from mechanisms that do not involve tRNAs. For instance, in Bacillus sphaericus, D-cysteine as well as L-cysteine behave as substrates of cystathionine -synthase (42). As a result, excess D- or L-cysteine triggers consumption of homoserine at the expense of threonine biosynthesis. Cysteine growth-inhibitory effects on E. coli cells were also reported to be mediated by an inhibition of threonine deaminase and the resulting starvation for isoleucine, leucine, and valine (22).

Although the toxicity indices associated with D-Glu, D-Thr, D-Lys, D-Val, and D-Cys did not vary upon inactivation of the dtd gene, the possibility that these D-amino acids can also be esterified to their corresponding tRNAs by E. coli synthetases cannot be completely excluded. Indeed, as discussed above in the case of D-phenylalanine, the inhibitory effect on E. coli growth of a given D-aminoacyl-tRNA may be dominated by other toxic mechanisms and, consequently, be not detectable in our experiments. Another possibility would be that tRNAs esterified with D-Glu, D-Thr, D-Lys, D-Val, and D-Cys are not substrates of D-Tyr-tRNA^Tyr deacylase.

Actually, D-valine binding to valyl-tRNA synthetase was reported by Owens and Bell (43, 44). However, the same authors observed stereospecificity in valyl-adenylate formation. Additional evidence against an esterification of the D-enantiomer to tRNA^Val was gained by Calendar and Berg (28) and Hélène et al. (45). At this stage, one must be aware of the difficulty to conclusively measure very slow rates of D-aminoacylation (10³ to 10⁴ s¹) without available radiolabeled D-amino acid. Moreover, a few aminoacyl-tRNA synthetases, including valyl-tRNA synthetase, have the capacity to promote hydrolysis of their misproducts through efficient proofreading mechanisms. Therefore, edition of D-aminoacyl-tRNAs by the synthetases themselves might also account for the difficulty to evidence the mischarged products. In the case of E. coli lysyl-tRNA synthetase, non-radioactive D-lysine was shown to significantly compete with L-[¹⁴C]lysine incorporation onto tRNA. Nevertheless, this inhibition can equally well be explained by non-radioactive L-lysine contaminating the D-lysine sample (46).

The screening enables us to also detect toxicity of D-Asn, D-His, D-Ile, D-Leu, and D-Met. D-Met can be suspected to be incorporated in cell walls, like D-Phe (23, 25, 26). The origin of the toxicity of D-histidine is unknown. However, it should not involve D-His-tRNA^His formation since histidyl-tRNA synthetase activity from Salmonella typhimurium was shown insensitive by close to 100% to the addition of up to 5 mM concentration of the D-enantiomer of histidine (47). In the cases of D-isoleucine and D-leucine, D-aminoacyl-adenylate formation by the corresponding E. coli synthetases was <1%, as compared with the reactions with the L-enantiomers (48). Moreover, E. coli isoleucyl-tRNA synthetase was shown to be specific for the L-enantiomer in ultracentrifuge studies on the binding of aliphatic amino acids (49, 50).

Comparison of E. coli and S. cerevisiae Sensitivities to D-Amino Acids-- As already shown (29), the toxicity of D-tyrosine toward yeast depends on the presence of a functional DTD1 allele. In the present study, the toxicity of the other D-amino acids was explored and inactivation of DTD1 was found to also exacerbate the sensitivity to D-leucine. This behavior may reflect the capacity of leucyl-tRNA synthetase to accept D-leucine as substrate. However, the sensitivity of E. coli growth to D-leucine did not vary upon disruption of the dtd gene. Such a difference between the two cells suggests a distinct specificity of the eubacterial leucyl-tRNA synthetase as compared with that of the fungal one. In a same manner, variations between the specificities of the bacterial and fungal synthetases may be invoked to explain the different behaviors of serine, tryptophan, glutamine, and aspartate in the absence of either the dtd or the DTD1 genes.

However, differences between the two cells may result from many other causes, as we shall now discuss. E. coli growth as well as that of yeast were not affected by the presence of D-proline. On the other hand, D-alanine and D-arginine slowed down yeast growth, not that of E. coli. D-Asparagine and D-isoleucine slightly affected E. coli growth but not that of yeast. Such apparent discrepancies in behaviors between the two cells may reflect the different metabolic pathways susceptible to play with D-amino acids in each case. For instance, in E. coli, D-alanine and D-glutamate naturally occur as products of specific racemases and contribute to cell wall biosynthesis. In E. coli K12 cells grown in minimal glucose medium, concentrations of these two D-amino acids are 0.5 and 1 mM, respectively (51). However, when added to the growth medium at a 25 mM concentration, D-glutamate slows down E. coli growth, while D-alanine does not. The lower sensitivity to D-alanine is possibly explained for by the occurrence of a bacterial D-amino acid dehydrogenase, which favors the transformation of amino acids with a hydrophobic character like D-Ala, D-Met, or D-Phe (52-54).

Transport systems of D-amino acids also are different in E. coli and in yeast. In E. coli, the permease produced by the aroP gene favors the import of aromatic amino acids. Upon deprivation of L-amino acids in the growth medium, this permease actively transports aromatic D-amino acids (55). In yeast, the import of both L- and D-amino acids involves the product of GAP1, a general permease with a broad specificity (21, 56).

Finally, in the yeast cytoplasm, D-amino acids are transformed by an -N-acetyltransferase (57). N-Acetylated amino acids are then believed to counter-react against further uptake of D-amino acids (21). The specificity of this acetylase is broad. However, its efficiency in the transformation of D-Pro, D-Asp, and D-Glu is relatively low (57, 58).

Broad Specificity of E. coli D-Tyr-tRNA Deacylase-- The broad specificity of E. coli D-Tyr-tRNA deacylase was early observed in vitro by Calendar and Berg (28). According to the present study, the deacylase recognizes very different D-aminoacyl moieties like the acidic aspartate or the bulky aromatic tryptophan. However, the deacylase appears not to simply behave as a D-amino acid esterase. Calendar and Berg showed that D-Tyr-adenosine was not hydrolyzed by the deacylase (28). In contrast, a D-Tyr-esterified oligonucleotide produced by RNase T1 digestion was a substrate. In the 19-mer oligonucleotide produced from tRNA^Tyr, the deacylase possibly recognizes a part of the acceptor stem of tRNA, for instance the 5'-CCA-3' triplet common to all tRNAs. On the side of the amino acid moiety, the deacylase would only distinguish the stereoisomeric character of the C. In agreement with this idea, glycyl-tRNA obtained in vitro was a substrate of the E. coli deacylase (28).

General Remarks-- As already discussed (29), dtd-like genes are widely distributed in the living world and deacylase homologs are expected to occur in many bacteria, as well as in yeasts, nematodes, higher plants, mouse, and man. The only exceptions are archaebacteria, parasitic auxotrophic bacteria, and prototrophic cyanobacteria.

In the case of auxotrophic bacteria, the lack of many biosynthetic pathways possibly results in especially low levels of endogenous D-amino acids. As a consequence, deacylase activity would be useless in such bacteria. In other cells, D-aminoacyl-tRNA deacylase activity would be necessary to counter-react against the harmful effect of D-amino acid transfer onto tRNA. The set of incorporated D-amino acids may vary from one type of cell to another. However, in each cell, one single species of deacylase should be enough to hydrolyze any D-aminoacyl-tRNA molecule.

A question to eventually address is why cells have maintained dtd-like genes rather than evolved through the selection of more specific aminoacyl-tRNA synthetases. Possibly, the primitive cell indifferently incorporated L- and D-amino acids into polypeptides. At this stage, acquisition of a general D-aminoacyl-tRNA deacylase activity could have helped the cell to shift protein synthesis in the L-amino acid world. According to this scenario, further evolution of cells implies an improvement of the specificity of the synthetases toward L-amino acids, and the progressive loss of a no more useful deacylase gene. Cyanobacteria and archaebacteria may correspond to such cells.

	ACKNOWLEDGEMENTS

We are grateful to Dr. J. Chen and Dr. A. Brevet for the construction of the tRNA^Asp gene, to Prof. C. Yanofsky for the gift of plasmid pCH17, and to Dr. G. Eriani for providing us with plasmid pBSaspS.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 33-1-69-33-41-81; Fax: 33-1-69-33-30-13; E-mail: plateau@coli.polytechnique.fr.

Published, JBC Papers in Press, July 28, 2000, DOI 10.1074/jbc.M005166200

	ABBREVIATIONS

The abbreviation used is: HPLC, high performance liquid chromatography.

	REFERENCES

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