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The aminoacylation of transfer ribonucleic acid. Recognition of methionine by Escherichia coli methionyl-transfer ribonucleic acid synthetase.

ABSTRACT: The mechanism of the recognition of methionine by Escherichia coli methionyl-tRNA synthetase was examined by a kinetic study of the recognition of methionine analogues in the ATP-PPi exchange reaction and the tRNA-aminoacylation reaction. The results show that the recognition mechanism consists of three parts: (1) the recognition of the size, shape and chemical nature of the amino acid side chain at the methionine-binding stage of the reaction; (2) the recognition of the length of the side chain at the stage of aminoacyl-adenylate complex-formation; (3) the recognition of the sulphur atom in the side chain at the stage of methionyl-tRNA formation. It is proposed that the sulphur atom interacts with the enzyme to induce a conformational change. A model of the active site incorporating the mechanism of methionine recognition is presented.

SUBMITTER: Old JM

PROVIDER: S-EPMC1164909 | biostudies-other | 1977 Aug

REPOSITORIES: biostudies-other

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Purification and properties of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli.

Project description:1. Methionyl-t-RNA synthetase (where t-RNA denotes ;soluble' or transfer RNA) has been purified to apparent homogeneity from a ribonuclease I-free strain of Escherichia coli. Polyacrylamide-gel electrophoresis of the final product revealed a single band. The purified enzyme catalyses the exchange of 450mumoles of pyrophosphate into ATP/mg. in 15min. at 37 degrees . 2. Methionyl-t-RNA synthetase is specific for the l-isomer of methionine, but appears to catalyse the methionylation of two distinct species of t-RNA, both of which are specific for methionine, but only one of which may be subsequently formylated. 3. The Michaelis constant for l-methionine is 2x10(-4)m in the ATP-PP(i) exchange assay and 2x10(-5)m for the acylation of t-RNA. 4. Gel filtration of both crude and highly purified preparations of methionyl-t-RNA synthetase on Sephadex G-200 indicates that the active species of enzyme has a molecular weight of about 190000. The amino acid composition of the enzyme is similar to those reported for the isoleucine and tyrosine enzymes from E. coli.

| S-EPMC1198268 | biostudies-other

Sub-unit structure and specificity of methionyl-transfer-ribonucleic acid synthetase from Escherichia coli.

Project description:1. The purification of methionyl-transfer-RNA synthetase from Escherichia coli by a modified technique gives a 16% yield of a protein that appears homogeneous by the criteria of disc gel electrophoresis, ultracentrifugation and end-group analysis. 2. The molecular weight is 96000 and the protein consists of two sub-units of 48000, which appear to be identical. The amino acid composition and thiol content are reported. 3. Kinetic data are reported for analogues of methionine and for pure t-RNA(F) and t-RNA(M), which are respectively the methionine transfer RNA that can exist in the formylmethionyl form and the one that can exist only in the methionyl form. The enzyme binds and acylates both species of transfer RNA identically.

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Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Escherichia coli by pseudomonic acid.

Project description:The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.

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A comparison of purified valyl-transfer ribonucleic acid synthetase from Bacillus stearothermophilus and from Escherichia coli.

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

Project description: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.

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Redesigning methionyl-tRNA synthetase for β-methionine activity with adaptive landscape flattening and experiments.

Project description:Amino acids (AAs) with a noncanonical backbone would be a valuable tool for protein engineering, enabling new structural motifs and building blocks. To incorporate them into an expanded genetic code, the first, key step is to obtain an appropriate aminoacyl-tRNA synthetase. Currently, directed evolution is not available to optimize AAs with noncanonical backbones, since an appropriate selective pressure has not been discovered. Computational protein design (CPD) is an alternative. We used a new CPD method to redesign MetRS and increase its activity towards β-Met, which has an extra backbone methylene. The new method considered a few active site positions for design and used a Monte Carlo exploration of the corresponding sequence space. During the exploration, a bias energy was adaptively learned, such that the free energy landscape of the apo enzyme was flattened. Enzyme variants could then be sampled, in the presence of the ligand and the bias energy, according to their β-Met binding affinities. Eighteen predicted variants were chosen for experimental testing; 10 exhibited detectable activity for β-Met adenylation. Top predicted hits were characterized experimentally in detail. Dissociation constants, catalytic rates, and Michaelis constants for both α-Met and β-Met were measured. The best mutant retained a preference for α-Met over β-Met; however, the preference was reduced, compared to the wildtype, by a factor of 29. For this mutant, high resolution crystal structures were obtained in complex with both α-Met and β-Met, indicating that the predicted, active conformation of β-Met in the active site was retained.

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Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation.

Project description:Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.

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A recurrent general RNA binding domain appended to plant methionyl-tRNA synthetase acts as a cis-acting cofactor for aminoacylation.

Project description:The cDNA encoding rice methionyl-tRNA synthetase was isolated. The protein exhibited a C-terminal polypeptide appended to a classical MetRS domain. This supplementary domain is related to endothelial monocyte activating polypeptide II (EMAPII), a cytokine produced in mammals after cleavage of p43, a component of the multisynthetase complex. It is also related to Arc1p and Trbp111, two tRNA binding proteins. We expressed rice MetRS and a derivative with a deletion of its EMAPII-like domain. Band-shift analysis showed that this extra-domain provides MetRS with non-specific tRNA binding properties. The EMAPII-like domain contributed a 10-fold decrease in K:(M) for tRNA in the aminoacylation reaction catalyzed by the native enzyme, as compared with the C-terminally truncated MetRS. Consequently, the EMAPII domain provides MetRS with a better catalytic efficiency at the free tRNA concentration prevailing in vivo. This domain binds the acceptor minihelix of tRNA(Met) and facilitates its aminoacylation. These results suggest that the EMAPII module could be a relic of an ancient tRNA binding domain that was incorporated into primordial synthetases for aminoacylation of RNA minihelices taken as the ancestor of modern tRNA.

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