How methionyl-tRNA synthetase creates its amino acid recognition pocket upon l-methionine binding

Serre, Laurence; Verdon, Grégory; Choinowski, Thomas; Hervouet, Nadège; Risler, Jean‐Loup; Zelwer, C.

doi:10.1006/jmbi.2001.4408

Cited by 79 publications

(76 citation statements)

References 48 publications

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“…We proposed that this feature of the side-chain electronic structure might contribute to activation of 3 by MetRS. Indeed, the importance of electron density at the ␦ position has been supported by the observation that the thioether of the bound methionine substrate forms hydrogen bonds with both the side chain of Tyr-260 and the backbone NH of Leu-13 in the active site of the enzyme (28).…”

Section: Resultsmentioning

confidence: 99%

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

Kiick

Saxon

Tirrell

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

859

741

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The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

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Section: Resultsmentioning

confidence: 99%

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

Kiick

Saxon

Tirrell

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

859

741

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“…To ensure complete coverage of the saturation mutagenesis library, we created a library of lower diversity by reducing the number of randomized sites from four to three, while increasing the number of clones that carry the library by optimizing ligation and transformation conditions. Examination of the crystal structure of Met-bound E. coli MetRS (22) reveals that the sulfur atom of Met is recognized through hydrogen bonds with O of Y260 and the backbone amide of L13. (Fig.…”

Section: Resultsmentioning

confidence: 99%

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Tanrikulu

Schmitt

Mechulam

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

112

133

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Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.click chemistry ͉ high-throughput screening ͉ protein engineering

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“…Attempts to obtain crystals of the S. aureus MetS enzyme have failed, but crystallographic studies on E. coli MetG have been more successful, both in native form and as a complex with methionine on April 8, 2019 by guest http://aac.asm.org/ (37,45). The hydrophobic binding pocket consists of 10 amino acids surrounding the L-methionine, whose amine group is hydrogen bonded to the carboxyl group of Asp52 and the carbonyl oxygen atom of Leu13 (45).…”

Section: Discussionmentioning

confidence: 99%

Mode of Action and Biochemical Characterization of REP8839, a Novel Inhibitor of Methionyl-tRNA Synthetase

Ochsner

Young

Stone

et al. 2005

Antimicrob Agents Chemother

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Aminoacyl-tRNA synthetases have attracted interest as essential and novel targets involved in bacterial protein synthesis. REP8839 is a potent inhibitor of MetS, the methionyl-tRNA synthetase in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA), and in Streptococcus pyogenes. The biochemical activity of REP8839 was shown by specific inhibition of purified S. aureus MetS (50% inhibitory concentration, <1.9 nM). Target specificity was confirmed by overexpression of the metS gene in S. aureus, resulting in an eightfold increase in the MIC for REP8839. Macromolecular synthesis assays in the presence of REP8839 demonstrated a dose-dependent inhibition of protein synthesis and RNA synthesis in S. pneumoniae R6, but only protein synthesis was affected in an isogenic rel mutant deficient in the stringent response. Strains with reduced susceptibility to REP8839 were generated by selection of strains with spontaneous mutations and through serial passages. Point mutations within the metS gene were mapped, leading to a total of 23 different amino acid substitutions within MetS that were located around the modeled active site. The most frequent MetS mutations were I57N, leading to a shift in the MIC from 0.06 g/ml to 4 g/ml, and G54S, resulting in a MIC of 32 g/ml that was associated with a reduced growth rate. The mutation prevention concentration was 32 g/ml in four S. aureus strains (methicillin-sensitive S. aureus and MRSA), which is well below the drug concentration of 2% (20,000 g/ml) in a topical formulation. In conclusion, we demonstrate by biochemical, physiologic, and genetic mode-of-action studies that REP8839 exerts its antibacterial activity through specific inhibition of MetS, a novel target.

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How methionyl-tRNA synthetase creates its amino acid recognition pocket upon l-methionine binding

Cited by 79 publications

References 48 publications

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Mode of Action and Biochemical Characterization of REP8839, a Novel Inhibitor of Methionyl-tRNA Synthetase

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