Identification and Characterization of a Chemical Compound that Inhibits Methionyl-tRNA Synthetase from Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen problematic in causing nosocomial infections and is highly susceptible to development of resistance to multiple antibiotics. The gene encoding methionyl-tRNA synthetase (MetRS) from P. aeruginosa was cloned and the resulting protein characterized. METHODS: MetRS was kinetically evaluated and the KM for its three substrates, methionine, ATP and tRNAMet were determined to be 35, 515, and 29 µM, respectively. P. aeruginosaMetRS was used to screen two chemical compound libraries containing 1690 individual compounds. RESULTS: A natural product compound (BM01C11) was identified that inhibited the aminoacylation function. The compound inhibited P. aeruginosa MetRS with an IC50 of 70 µM. The minimum inhibitory concentration (MIC) of BM01C11 was determined against nine clinically relevant bacterial strains, including efflux pump mutants and hypersensitive strains of P. aeruginosa and E. coli. The MIC against the hypersensitive strain of P. aeruginosa was 16 µg/ml. However, the compound was not effective against the wild-type and efflux pump mutant strains, indicating that efflux may not be responsible for the lack of activity against the wild-type strains. When tested in human cell cultures, the cytotoxicity concentration (CC50) was observed to be 30 µg/ml. The compound did not compete with methionine or ATP for binding MetRS, indicating that the mechanism of action of the compound likely occurs outside the active site of aminoacylation. CONCLUSION: An inhibitor of P. aeruginosa MetRS, BM01C11, was identified as a flavonoid compound named isopomiferin. Isopomiferin inhibited the enzymatic activity of MetRS and displayed broad spectrum antibacterial activity. These studies indicate that isopomiferin may be amenable to development as a therapeutic for bacterial infections.

In situ kinetic trapping reveals a fingerprint of reversible protein thiol oxidation in the mitochondrial matrix.

Reactive oxygen species (ROS) are released at the mitochondrial inner membrane by the electron transport chain (ETC). Increasing evidence suggests that mitochondrial H2O2 acts as a signaling molecule and participates in the (feedback) regulation of mitochondrial activity and turnover. It seems likely that key mitochondrial components contain redox-sensitive thiols that help to adapt protein function to changes in electron flow. However, the identity of most redox-regulated mitochondrial proteins remains to be defined. Thioredoxin 2 (Trx2) is the major protein-thiol-reducing oxidoreductase in the mitochondrial matrix. We used in situ mechanism-based kinetic trapping to identify disulfide-exchange interactions of Trx2 within functional mitochondria of intact cells. Mass spectrometry successfully identified known and suspected Trx2 target proteins and, in addition, revealed a set of new candidate target proteins. Our results suggest that the mitochondrial protein biosynthesis machinery is a major target of ETC-derived ROS. In particular, we identified mitochondrial methionyl-tRNA synthetase (mtMetRS) as one of the most prominent Trx2 target proteins. We show that an increase in ETC-derived oxidants leads to an increase in mtMetRS oxidation in intact cells. In conclusion, we find that in situ kinetic trapping provides starting points for future functional studies of intramitochondrial redox regulation.

Crystallization of Mycobacterium smegmatis methionyl-tRNA synthetase in the presence of methionine and adenosine.

Methionyl-tRNA synthetase (MetRS) from Mycobacterium smegmatis was recombinantly expressed in Escherichia coli and purified using Ni(2+)-affinity and size-exclusion chromatography. Crystals formed readily in the presence of the ligands methionine and adenosine. These two ligands are components of an intermediate in the two-step catalytic mechanism of MetRS. The crystals were produced using the vapour-diffusion method and a full data set to 2.1 A resolution was collected from a single crystal. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 155.9, b = 138.9, c = 123.3 A, beta = 124.8 degrees . The presence of three molecules in the asymmetric unit corresponded to a solvent content of 60% and a Matthews coefficient of 3.1 A(3) Da(-1). Structure determination is in progress.

An operational RNA code for faithful assignment of AUG triplets to methionine.

The assignment of AUG codons to methionine remains a central question of the evolution of the genetic code. We have unveiled a strategy for the discrimination among tRNAs containing CAU (AUG-decoding) anticodons. Mycoplasma penetrans methionyl-tRNA synthetase can directly differentiate between tRNA(Ile)(CAU) and tRNA(Met)(CAU) transcripts (a recognition normally achieved through the modification of anticodon bases). This discrimination mechanism is based only on interactions with the acceptor stems of tRNA(Ile)(CAU) and tRNA(Met)(CAU). Thus, in certain species, the fidelity of translation of methionine codons requires a discrimination mechanism that is independent of the information contained in the anticodon.

Reconstitution of yeast translation initiation.

To facilitate the mechanistic dissection of eukaryotic translation initiation we have reconstituted the steps of this process using purified Saccharomyces cerevisiae components. This system provides a bridge between biochemical studies in vitro and powerful yeast genetic techniques, and complements existing reconstituted mammalian translation systems (Benne and Hershey, 1978; Pestova and Hellen, 2000; Pestova et al., 1998; Trachsel et al., 1977). The following describes methods for synthesizing and purifying the components of the yeast initiation system and assays useful for its characterization.

Characterization of the human mitochondrial methionyl-tRNA synthetase.

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn(2+) binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K(M) and k(cat) for aminoacylation of E. coli initiator tRNA(f)(Met) are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA(Met) (mtRNA(Met)) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA(Met) and of an in vitro transcript of the bovine mtRNA(Met) have also been investigated.

Evidence for breaking domain-domain functional communication in a synthetase-tRNA complex.

We report here evidence for mutations that break domain-domain functional communication in a synthetase-tRNA complex. Each synthetase is roughly divided into two major domains that are paralleled by the two arms of the L-shaped tRNA structure. The active-site-containing domain interacts with the acceptor arm of the tRNA. The second domain frequently interacts with the anticodon-containing arm. By an induced-fit mechanism, contacts with the anticodon can activate formation of a robust transition state at a site over 70 A away. This induced-fit-based activation is thought to occur through domain-domain signaling and is seen by the enhancement of aminoacylation of the anticodon-containing full tRNA versus a substrate based on the acceptor arm alone. Here we describe a rationally designed mutant methionyl-tRNA synthetase containing two point substitutions at sites that potentially link an anticodon-binding motif to the catalytic domain. The double mutation had no effect on interactions with either the isolated acceptor arm or the anticodon stem-loop. In contrast to interactions with the separate pieces, the mutant enzyme was severely impaired for binding the native tRNA and lost much of its ability to enhance the rate of charging of the full tRNA over that of a substrate based on the acceptor arm alone. We propose that these residues are part of a network for facilitating domain-domain communication for formation of an active synthetase-tRNA complex by induced fit.

A site in the dinucleotide-fold domain contributes to the accuracy of tRNA selection by Escherichia coli methionyl-tRNA synthetase.

Interactions of specific amino acid residues of the carboxyl-terminal domain of MetRS with the CAU anticodon of tRNAMet assure accurate and efficient aminoacylation. The substitution of one such residue, Trp461 by Phe, impairs the binding of cognate tRNA, but enhances the binding of noncognate tRNAs, particularly those containing G at the wobble position. However, the enhanced binding of noncognate tRNAs is not accompanied by the increased aminoacylation of these tRNAs. A genetic screening procedure was designed to isolate methionyl-tRNA synthetase mutants which were able to aminoacylate a GGU (threonine) anticodon derivative of tRNAfMet. One such mutant, obtained from W461F MetRS, had an Ile29 to Thr substitution in helix A located in the amino-terminal dinucleotide-fold domain that forms the site for amino acid activation. Analysis of the catalytic properties of the I29T/W461F enzyme indicates that the mutation in helix A of the dinucleotide-fold domain affects kcat for aminoacylation of tRNAs having a GGU threonine anticodon. Interactions with cognate tRNAfMet (CAU), as well as with methionine and ATP were not affected by the Ile29 to Thr substitution. We conclude that the I29T substitution leads to a slight adjustment of the alignment of the CCA stem of noncognate tRNAs (GGU) in the catalytic domain of the enzyme, reflected in the increase in kcat, which also allows mischarging in vivo. A function of Ile29 is therefore to minimize the mischarging of tRNAThr (GGU) by methionyl-tRNA synthetase. The methods described here provide useful tools for examining the mechanisms of tRNA selection by aminoacyl-tRNA synthetases.

A single gene of chloroplast origin codes for mitochondrial and chloroplastic methionyl-tRNA synthetase in Arabidopsis thaliana.

One-fifth of the tRNAs used in plant mitochondrial translation is coded for by chloroplast-derived tRNA genes. To understand how aminoacyl-tRNA synthetases have adapted to the presence of these tRNAs in mitochondria, we have cloned an Arabidopsis thaliana cDNA coding for a methionyl-tRNA synthetase. This enzyme was chosen because chloroplast-like elongator tRNAMet genes have been described in several plant species, including A. thaliana. We demonstrate here that the isolated cDNA codes for both the chloroplastic and the mitochondrial methionyl-tRNA synthetase (MetRS). The protein is transported into isolated chloroplasts and mitochondria and is processed to its mature form in both organelles. Transient expression assays using the green fluorescent protein demonstrated that the N-terminal region of the MetRS is sufficient to address the protein to both chloroplasts and mitochondria. Moreover, characterization of MetRS activities from mitochondria and chloroplasts of pea showed that only one MetRS activity exists in each organelle and that both are indistinguishable by their behavior on ion exchange and hydrophobic chromatographies. The high degree of sequence similarity between A. thaliana and Synechocystis MetRS strongly suggests that the A. thaliana MetRS gene described here is of chloroplast origin.

Arginine-395 is required for efficient in vivo and in vitro aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase.

We have previously shown that the anticodon of methionine tRNAs contains the major recognition site required for aminoacylation of tRNAs by Escherichia coli methionyl-tRNA synthetase (MetRS) and have located part of the anticodon binding domain on the enzyme at a site close to Trp461 [Schulman, L. H., & Pelka, H. (1988) Science 242, 765-768; Ghosh, G., Pelka, H., & Schulman, L.H. (1990) Biochemistry 29, 2220-2225]. In order to gain information about other possible sites of contact between MetRS and its tRNA substrates, we have examined the effects of mutations at a series of positively charged residues on the surface of the C-terminal domain of the enzyme. Conversion of Arg356, Arg366, Arg380, or Arg453 to Gln had little or no effect on enzyme activity. Similarly, conversion of Lys402 or Lys439 to Asn failed to significantly alter aminoacylation activity. Conversion of Arg380 to Ala or Arg442 to Gln produced a 5-fold reduction in kcat/Km for aminoacylation of tRNAfMet, with no effect on methionine activation, indicating a possible minor role for these residues in interaction of the enzyme with the tRNA substrate. In contrast, mutation of a phylogenetically conserved residue, Arg395, to Gln increased the Km for aminoacylation of tRNAfMet about 30-fold and reduced kcat/Km by 25,000-fold. The mutant enzyme was also shown to be highly defective by its inability to complement a strain of E. coli having an altered chromosomal MetRS gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of methionine by Escherichia coli methionyl-tRNA synthetase.

In the present work, we have examined the function of three amino acid residues in the active site of Escherichia coli methionyl-tRNA synthetase (MetRS) in substrate binding and catalysis using site-directed mutagenesis. Conversion of Asp52 to Ala resulted in a 10,000-fold decrease in the rate of ATP-PPi exchange catalyzed by MetRS with little or no effect on the Km's for methionine or ATP or on the Km for the cognate tRNA in the aminoacylation reaction. Substitution of the side chain of Arg233 with that of Gln resulted in a 25-fold increase in the Km for methionine and a 2000-fold decrease in kcat for ATP-PPi exchange, with no change in the Km for ATP or tRNA. These results indicate that Asp52 and Arg233 play important roles in stabilization of the transition state for methionyl adenylate formation, possibly directly interacting with complementary charged groups (ammonium and carboxyl) on the bound amino acid. Primary sequence comparisons of class I aminoacyl-tRNA synthetases show that all but one member of this group of enzymes has an aspartic acid residue at the site corresponding to Asp52 in MetRS. The synthetases most closely related to MetRS (including those specific for Ile, Leu, and Val) also have a conserved arginine residue at the position corresponding to Arg233, suggesting that these conserved amino acids may play analogous roles in the activation reaction catalyzed by each of these enzymes. Trp305 is located in a pocket deep within the active site of MetRS that has been postulated to form the binding cleft for the methionine side chain.(ABSTRACT TRUNCATED AT 250 WORDS)

An anticodon change switches the identity of E. coli tRNA(mMet) from methionine to threonine.

Recent evidence indicates that the anticodon may often play a crucial role in selection of tRNAs by aminoacyl-tRNA synthetases. In order to quantitate the contribution of the anticodon to discrimination between cognate and noncognate tRNAs by E. coli threonyl-tRNA synthetase, derivatives of the E. coli elongator methionine tRNA (tRNA(mMet)) containing wild type and threonine anticodons have been synthesized in vitro and assayed for threonine acceptor activity. Substitution of the threonine anticodon GGU for the methionine anticodon CAU increased the threonine acceptor activity of tRNA(mMet) by four orders of magnitude while reducing methionine acceptor activity by an even greater amount. These results indicate that the anticodon is the major element which determines the identity of both threonine and methionine tRNAs.

[Methionyl-tRna synthetase from wheat germ. Effect of an endogenous protease and correlations between structural characteristics and catalytic properties]. / Méthionyl-tRNA synthétase du germe de blé. Effet d'une protéase endogène et corrélations entre caractéristiques structurales et propriétés catalytiques.

Methionyl-tRNA synthetase (MetRS) has been described as a free monomeric or oligomeric enzyme; or included in a multienzyme complex. Moreover, on limited tryptic digestion, it can generate shorter forms. So, when purified from wheat-germ lysate, the possible presence of proteases able to hydrolyse this enzyme was investigated. When extraction was performed with sulfhydryl-blocking reagents, an active monomeric MetRS of Mr 105,000 was purified. This enzyme form was identical to the structure exhibiting methionyl-tRNA synthetase activity in multienzyme complexes. Without this inhibitor, MetRS was purified as an active dimeric form of Mr 165,000 with identical subunits of Mr 82,000. A protease inhibited by sulfhydryl-blocking reagents and included in a complex of Mr 2.10(6) was isolated from this wheat-germ lysate. This protease was able to hydrolyse different proteins (albumin, casein), but was without activity for a trypsin substrate, such as N-alpha-benzoyl-DL-arginine p-nitroanilide. When added to a solution of Mr-105,000 MetRS, it yielded an inactive peptide of Mr 20,000, containing numerous charged amino acids and a protein of Mr 82,000, able to give an active dimeric enzyme of Mr 165,000. Amino acid analysis of this last form, indicated an identical structure with the active dimeric MetRS of Mr 165,000, purified in the absence of protease inhibitors. Moreover, the affinity for methionine was the same for the monomeric enzyme of Mr 105,000 and the dimeric form of Mr 165,000, probably because proteolysis did not affect the catalytic domain. When enzymic activity of the proteolyzed form (Mr 2 x 82,000) was studied versus enzyme concentration, a decrease in specific activity, at low concentrations, was seen. This phenomenon was analysed on the basis of the existence of an equilibrium between an active dimer and two inactive monomers. With the active monomeric form of Mr 105,000, no change in specific activity with decreasing enzyme concentration occurred.

Purification of the yeast mitochondrial methionyl-tRNA synthetase. Common and distinctive features of the cytoplasmic and mitochondrial isoenzymes.

Yeast-mitochondrial methionyl-tRNA synthetase was purified 1060-fold from mitochondrial matrix proteins of Saccharomyces cerevisiae using a four-step procedure based on affinity chromatography (heparin-Ultrogel, tRNA(Met)-Sepharose, Agarose-hexyl-AMP) to yield to a single polypeptide of high specific activity (1800 U/mg). Like the cytoplasmic methionyl-tRNA synthetase (Mr 85,000), the mitochondrial isoenzyme is a monomer, but of significantly smaller polypeptide size (Mr 65,000). In contrast, the corresponding enzyme of Escherichia coli is a dimer (Mr 152,000) made up of identical subunits. The measured affinity constants of the purified mitochondrial enzyme for methionine and tRNA(Met) are similar to those of the cytoplasmic isoenzyme. However, the two yeast enzymes exhibit clearly different patterns of aminoacylation of heterologous yeast and E. coli tRNA(Met). Furthermore, polyclonal antibodies raised against the two proteins did not show any cross-reactivity by inhibition of enzymatic activity and by the highly sensitive immunoblotting technique, indicating that the two enzymes share little, if any, common antigenic determinants. Taken together, our results further support the belief that the yeast mitochondrial and cytoplasmic methionyl-tRNA synthetases are different proteins coded for by two distinct nuclear genes. Like the yeast cytoplasmic aminoacyl-tRNA synthetases, the mitochondrial enzymes displayed affinity for immobilized heparin. This distinguishes them from the corresponding enzymes of E. coli. Such an unexpected property of the mitochondrial enzymes suggests that they have acquired during evolution a domain for binding to negatively charged cellular components.

Purification and NMR studies of [methyl-13C]methionine-labeled truncated methionyl-tRNA synthetase.

A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed. With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase [Barker, D.G., Ebel, J.-P., Jakes, R., & Bruton, C.J. (1982) Eur. J. Biochem. 127, 449], can be obtained. The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases. A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies. With this procedure, truncated methionyl-tRNA synthetase labeled with [methyl-13C]methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase. Both carbon-13 and proton observe-heteronuclear detect NMR experiments were used to observe the 13C-enriched methyl resonances of the 17 methionine residues in the truncated synthetase. In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR. Titration of the synthetase, monitoring the chemical shifts of resonances B and M (Figure 3), with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Functions of isolated domains of methionyl-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8.

Methionyl-tRNA synthetase (MetRS, 2 X 75 kDa) was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB8. The polypeptide chain of MetRS was cleaved by limited digestion with trypsin into four domains: T1 (29 kDa), T2 (23 kDa), T3 (14.5 kDa), and T4 (7.5 kDa), which were aligned in that order. MetRS was also cleaved into similar fragments with a variety of other proteases. Domains T1, T2, T3, and T4 were isolated by column chromatography. "Tandem domain" T1-T2 (56 kDa) is fully active in the aminoacylation of tRNA and is further cleaved with trypsin into domains T1 and T2. Domain T1 is the smallest aminoacylation unit so far reported. Domain T2 (enzymatically inactive) interacts with tRNAMetf, as found by UV-induced cross-linking. Isolated domain T3 forms a dimer and is responsible for the dimer assembly of two protomers in MetRS. Domain T4 is a flexible tail of MetRS. These domains, in particular T1 and T2, will be important for detailed structure analyses in relation to aminoacylation activity.

Purification and characterization of the isoleucyl-tRNA synthetase component from the high molecular weight complex of sheep liver: a hydrophobic metalloprotein.

Native isoleucyl-tRNA synthetase and a structurally modified form of methionyl-tRNA synthetase were purified to homogeneity following trypsinolysis of the high molecular weight complex from sheep liver containing eight aminoacyl-tRNA synthetases. The correspondence between purified isoleucyl-tRNA synthetase and the previously unassigned polypeptide component of Mr 139 000 was established. It is shown that dissociation of this enzyme from the complex has no discernible effect on its kinetic parameters. Both isoleucyl- and methionyl-tRNA synthetases contain one zinc ion per polypeptide chain. In both cases, removal of the metal ion by chelating agents leads to an inactive apoenzyme. As the trypsin-modified methionyl-tRNA synthetase has lost the ability to associate with other components of the complex [Mirande, M., Kellermann, O., & Waller, J. P. (1982) J. Biol. Chem. 257, 11049-11055], the zinc ion is unlikely to be involved in complex formation. While native purified isoleucyl-tRNA synthetase displays hydrophobic properties, trypsin-modified methionyl-tRNA synthetase does not. It is suggested that the assembly of the amino-acyl-tRNA synthetase complex is mediated by hydrophobic domains present in these enzymes.

Generation of multiple forms of methionyl-tRNA synthetase from the multi-enzyme complex of mammalian aminoacyl-tRNA synthetases by endogenous proteolysis.

Methionyl-tRNA synthetase occurs free and as high-molecular-weight multi-enzyme complexes in rat liver. The free form is purified to near homogeneity by conventional column chromatography and affinity chromatography on tRNA-Sepharose. The native molecular weight of free methionyl-tRNA synthetase is 64 500, based on its sedimentation coefficient of 4.5 S and Stokes radius of 33 A. The free methionyl-tRNA synthetase apparently belongs to alpha-type subunit structure, since the subunit molecular weight is 68 000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Methionyl-tRNA synthetase is dissociated from the high-molecular-weight synthetase complex by controlled trypsinization, according to Kellermann, O., Viel, C. and Waller, J.P. (Eur. J. Biochem. 88 (1978) 197-204). The dissociated, free methionyl-tRNA synthetase is subsequently purified to near homogeneity. The subunit structure of dissociated methionyl-tRNA synthetase is identical to that of endogenous free methionyl-tRNA synthetase. Anti-serum raised against Mr 104 000 protein in the synthetase complex, specifically inhibited methionyl-tRNA synthetase in both the free and the high-molecular-weight forms to the same extent. These results suggest that the occurrence of multiple forms of methionyl-tRNA synthetases in mammalian cells may, in part, be due to proteolytic cleavage.

Methionyl-tRNA synthetase from Escherichia coli. Primary structure of the active crystallised tryptic fragment.

A 3300-base segment of Escherichia coli chromosomal DNA, cloned into pBR322, will complement a methionine auxotroph in which the lesion is a defective methionyl-tRNA synthetase with a much reduced affinity for methionine. Crude extracts of these transformants contain elevated levels of a protein which has a subunit molecular weight of 66 000, methionyl-tRNA synthetase aminoacylation activity in vitro and which cross-reacts with anti-(methionyl-tRNA synthetase) antibodies. This polypeptide is very slightly larger than the well-characterised and crystallised tryptic fragment of methionyl-tRNA synthetase. A DNA sequence of 1750 residues at one end of the cloned insert codes for a non-terminated open reading frame in which we can locate a large number of methionyl-tRNA synthetase tryptic and chymotryptic peptides. We have also sequenced 300 nucleotides upstream of this coding segment where we find a large invert repeat in the putative methionyl-tRNA synthetase promoter region.