Pierre Plateau

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

In Escherichia coli, tyrosyl-tRNA synthetase is known to esterify tRNATyr with tyrosine. Resulting d-Tyr-tRNATyr can be hydrolyzed by ad-Tyr-tRNATyr 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 encodingd-Tyr-tRNATyr 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-tRNAAspand d-Trp-tRNATrp by aspartyl-tRNA synthetase and tryptophanyl-tRNA synthetase, respectively, was establishedin vitro. Furthermore, the two d-aminoacylated tRNAs behaved as substrates of purified E. coli d-Tyr-tRNATyr deacylase. These results indicate that an unexpected high number of d-amino acids can impair the bacterium growth through the accumulation ofd-aminoacyl-tRNA molecules and thatd-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 producesd-Tyr-tRNATyr, and which, like E. coli, possesses a d-Tyr-tRNATyr deacylase activity. In this case, inhibition of growth by the various 19d-amino acids was followed on solid medium. Two isogenic strains containing or not the deacylase were compared. Toxic effects ofd-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 twod-aminoacyl-tRNAs are substrates of a samed-aminoacyl-tRNA deacylase.

Extracellular Production of Hydrogen Selenide Accounts for Thiol-assisted Toxicity of Selenite against Saccharomyces cerevisiae

Administration of selenium in humans has anticarcinogenic effects. However, the boundary between cancer-protecting and toxic levels of selenium is extremely narrow. The mechanisms of selenium toxicity need to be fully understood. In Saccharomyces cerevisiae, selenite in the millimolar range is well tolerated by cells. Here we show that the lethal dose of selenite is reduced to the micromolar range by the presence of thiols in the growth medium. Glutathione and selenite spontaneously react to produce several selenium-containing compounds (selenodiglutathione, glutathioselenol, hydrogen selenide, and elemental selenium) as well as reactive oxygen species. We studied which compounds in the reaction pathway between glutathione and sodium selenite are responsible for this toxicity. Involvement of selenodiglutathione, elemental selenium, or reactive oxygen species could be ruled out. In contrast, extracellular formation of hydrogen selenide can fully explain the exacerbation of selenite toxicity by thiols. Indeed, direct production of hydrogen selenide with d-cysteine desulfhydrase induces high mortality. Selenium uptake by S. cerevisiae is considerably enhanced in the presence of external thiols, most likely through internalization of hydrogen selenide. Finally, we discuss the possibility that selenium exerts its toxicity through consumption of intracellular reduced glutathione, thus leading to severe oxidative stress.

Solvent-peak-suppressed NMR: Correction of baseline distortions and use of strong-pulse excitation

Abstract Pulse excitation sequences which involve time delays lead to spectra suffering from so-called frequency-dependent phase shifts. The phenomenon is analyzed in a model case, and the above description is shown to be somewhat improper. Rather, the spectrum should be described as a sum of Lorentzian lines, each of which has its own, frequency-independent , phase shift. The analysis leads to procedures for correcting the baseline distortions of solvent-suppressed NMR based on the Redfield 2-1-4 sequence, or on analogous strong-pulse sequences which are described. Goals and methods of solvent-signal suppression are discussed in this context.

Functional characterization of the D-Tyr-tRNATyr deacylase from Escherichia coli.

The yihZ gene of Escherichia coli is shown to produce a deacylase activity capable of recycling misaminoacylated d-Tyr-tRNATyr. The reaction is specific and, under optimal in vitroconditions, proceeds at a rate of 6 s−1 with aK m value for the substrate equal to 1 μm. Cell growth is sensitive to interruption of theyihZ gene if d-tyrosine is added to minimal culture medium. Toxicity of exogenous d-tyrosine is exacerbated if, in addition to the disruption of yihZ, the gene of d-amino acid dehydrogenase (dadA) is also inactivated. Orthologs of the yihZ gene occur in many, but not all, bacteria. In support of the idea of a general role of thed-Tyr-tRNATyr deacylase function in the detoxification of cells, similar genes can be recognized inSaccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, mouse, and man.

The role of zinc in 5′,5′-diadenosine tetraphosphate production by aminoacyl-transfer RNA synthetases

SummaryAminoacyl-tRNA synthetases are capable of converting 5′-ATP into 5′,5′-diadenosine tetraphosphate. The reaction reflects the reversal of enzyme-bound aminoacyl-adenylate by ATP instead of PPi.In the case of a few prokaryotic as well as eukaryotic aminoacyl-tRNA synthetases, the initial rate of diadenosine tetraphosphate synthesis can be greatly enhanced upon adding small amounts of zinc. This observation enables us to establish a relationship between diadenosine tetraphosphate, a nucleotide possibly involved in controlling cell proliferation, and a metallic cofactor, which is believed to play a role in tumour growth.

Formation of d-Tyrosyl-tRNATyr Accounts for the Toxicity of d-Tyrosine toward Escherichia coli

d-Tyr-tRNATyr deacylase cleaves the ester bond between a tRNA molecule and a d-amino acid. In Escherichia coli, inactivation of the gene (dtd) encoding this deacylase increases the toxicity of several d-amino acids including d-tyrosine, d-tryptophan, and d-aspartic acid. Here, we demonstrate that, in a Δdtd cell grown in the presence of 2.4 mm d-tyrosine, ∼40% of the total tRNATyr pool is converted into d-Tyr-tRNATyr. No d-Tyr-tRNATyr is observed in dtd+ cells. In addition, we observe that overproduction of tRNATyr, tRNATrp, or tRNAAsp protects a Δdtd mutant strain against the toxic effect of d-tyrosine, d-tryptophan, or d-aspartic acid, respectively. In the case of d-tyrosine, we show that the protection is accounted for by an increase in the concentration of l-Tyr-tRNATyr proportional to that of overproduced tRNATyr. Altogether, these results indicate that, by accumulating in vivo, high amounts of d-Tyr-tRNATyr cause a starvation for l-Tyr-tRNATyr. The deacylase prevents the starvation by hydrolyzing d-Tyr-tRNATyr. Overproduction of tRNATyr also relieves the starvation by increasing the amount of cellular l-Tyr-tRNATyr available for translation.

Free-Energy Simulations and Experiments Reveal Long-Range Electrostatic Interactions and Substrate-Assisted Specificity in an Aminoacyl-tRNA Synthetase

Specific recognition of their cognate amino acid substrates by the aminoacyl‐tRNA synthetase enzymes is essential for the correct translation of the genetic code. For aspartyl‐tRNA synthetase (AspRS), electrostatic interactions are expected to play an important role, since its three substrates (aspartate, ATP, tRNA) are all electrically charged. We used molecular‐dynamics free‐energy simulations and experiments to compare the binding of the substrate Asp and its electrically neutral analogue Asn to AspRS. The preference for Asp is found to be very strong, with good agreement between simulations and experiment. The simulations reveal long‐range interactions that electrostatically couple the amino acid ligand, ATP, and its associated Mg2+ cations, a histidine side chain (His448) next to the amino acid ligand and a flexible loop that closes over the active site in response to amino acid binding. Closing this loop brings a negatively charged glutamate into the active site; this causes His448 to recruit a labile proton, which interacts favorably with Asp and accounts for most of the Asp/Asn discrimination. Cobinding of the second substrate, ATP, increases specificity for Asp further and makes the system robust towards removal of His448, which is mutated to a neutral amino acid in many organisms. Thus, AspRS specificity is assisted by a labile proton and a cosubstrate, and ATP acts as a mobile discriminator for specific Asp binding to AspRS. In asparaginyl‐tRNA synthetase, a close homologue of AspRS, a few binding‐pocket differences modify the charge balance so that asparagine binding predominates.

Sequence, overproduction and crystallization of aspartyl-tRNA synthetase from Thermus thermophilus: Implications for the structure of prokaryotic aspartyl-tRNA synthetases

The genes of aspartyl‐tRNA synthetase (AspRS) from two Thermus thermophilus strains VK.‐1 and HB8, have been cloned and sequenced. Their nucleotidic sequences code for the same protein which displays the three characteristic motifs of class II aminoacyl‐tRNA synthetases. This enzyme shows 50% identity with Escherichia coli AspRS, over the totality of the chain (580 amino acids). A comparison with the eukaryotic yeast cytoplasmic AspRS indicates the presence in the prokaryotic AspRS of an extra domain between motifs 2 and 3 much larger than in the eukaryotic ones. When its gene is under the control of the tac promoter of the expression vector pKK223‐3, the protein is efficiently overexpressed as a thermostable protein in E. coli. It can be further purified to homogeneity using a heat treatment followed by a single anion exchange chromatography. Single crystals of the pure protein, diffracting at least to 2.2 Å resolution (space group P212121, a = 61.4 Å, b = 156.1 Å, c = 177.3 Å) are routinely obtained. The same crystals have previously been described as crystals of threonyl‐tRNA synthetase [1].

Sodium Selenide Toxicity Is Mediated by O2-Dependent DNA Breaks

Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H2Se/HSe−/Se2−). Among the genes whose deletion caused hypresensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O2-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The •OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O2. Finally we showed that, in vivo, toxicity strictly depended on the presence of O2. Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O2-dependent radical-based mechanism.

Uptake of Selenite by Saccharomyces cerevisiae Involves the High and Low Affinity Orthophosphate Transporters

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low Pi concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity Pi transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low Pi medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both Pi transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.