Metabolism of d-Aminoacyl-tRNAs inEscherichia coli and Saccharomyces cerevisiae Cells

Abstract

Trpby 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-tRNATyr 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. 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)(18)(19)(20)(21)(22)(23)(24)(25)(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 indicateTyr 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 ...

The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

“…Considering the likelihood that multiple D-aa-tRNAs might be incorporated by a single elongating ribosome during the translation of a single protein, it is possible that the accumulation of translationally arrested ribosomes and/or of the truncated protein products that they produce could contribute significantly to the observed cytotoxicity of D-amino acids. Given that D-amino acid racemase enzymes are found in the brain (6), that D-amino acids can be aminoacylated onto tRNA by aaRS (8,9), and that tRNA misacylation has been linked to neurodegenerative disorders (36), the translation disorders that we report here provide a plausible molecular mechanism that warrants future consideration as a potential underlying cause for neurodegenerative disease.…”

“…eukaryotic cells results in the cytotoxicity of several D-amino acids that in the presence of dtd are typically nontoxic (9). This cytotoxicity has been attributed either to compromising the downstream function of proteins into which D-amino acids have been incorporated (34) or to depleting the pool of L-aa-tRNAs from cells via the misacylation of D-amino acids onto tRNAs (35).…”

“…First, improved incorporation of D-amino acids by the TM would be useful for protein engineering applications that seek to use the synthetic power of the ribosome to create novel polymers (4) as well as for mechanistic applications that seek to probe protein structure and folding with unnatural amino acids (5). Second, there is growing evidence that ribosomes may have to contend with D-aa-tRNAs in vivo: D-amino acids are synthesized by racemase enzymes (6) and can be found at high concentrations in cells (7), and a growing number of aa-tRNA synthetase (aaRS) enzymes exhibit the ability to misacylate tRNAs with D-amino acids (8,9). Consistent with this possibility, the D-aa-tRNA deacylase (DTD) enzyme is nearly universally conserved (10) and functions to remove D-amino acids that have been misacylated onto tRNA (8, 10).…”

Ammonium Scanning in an Enzyme Active Site

“…Kinetic experiments show, however, that AspRS does not strongly distinguish between the "left-handed" L-Asp and "right-handed" D-Asp stereoisomers. Indeed, D-Asp-tRNA Asp is produced at detectable levels by Escherichia coli AspRS (26), at a rate only 4,000 times lower than L-Asp-tRNA Asp . To preserve homochirality in vivo (27), editing (28) of D-Asp-tRNA Asp by a D-aminoacyl-tRNA deacylase is performed (26).…”

Genetic incorporation of d-lysine into diketoreductase in Escherichia coli cells

“…Consequently, this orthogonal pair was employed in the present study to explore the possibility of incorporating D-Lys into proteins. Meanwhile, although D-Tyrosyl-tRNA deacylase showed broad substrate specificity primarily responsible for the cleavage of the linkage between most D-amino acids and tRNA to avoid the incorporation of D-amino acids by ribosomal protein synthesis machinery, deletion of the dtd gene did not reduce the toxicity of E. coli K37 by various D-amino acids, including D-Lys, [8] suggesting that the hydrolytic activity of D-Tyrosyl-tRNA deacylase towards DLysyl-tRNA might be very weak. In addition, the absence of specific D-Lysyl-tRNA deacylase activity in E. coli further increases the possibility that D-Lysyl-tRNA could be stable in cells and thus permitting D-Lys to be incorporated into proteins.…”