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Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase.

ABSTRACT: The specificity of most aminoacyl-tRNA synthetases for an amino acid and cognate tRNA pair evolved before the divergence of the three domains of life. Glutaminyl-tRNA synthetase (GlnRS) evolved later and is derived from the archaeal-type nondiscriminating glutamyl-tRNA synthetase (GluRS), an enzyme with relaxed tRNA specificity capable of forming both Glu-tRNA(Glu) and Glu-tRNA(Gln). The archaea lack GlnRS and use a specialized amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) needed for protein synthesis. We show that the Methanothermobacter thermautotrophicus GluRS is active toward tRNA(Glu) and the two tRNA(Gln) isoacceptors the organism encodes, but with a significant catalytic preference for tRNA(Gln2)(CUG). The less active tRNA(Gln1)(UUG) responds to the less common CAA codon for Gln. From a biochemical characterization of M. thermautotrophicus GluRS variants, we found that the evolution of tRNA specificity in GlnRS could be recapitulated by converting the M. thermautotrophicus GluRS to a tRNA(Gln) specific enzyme, solely through the addition of an acceptor stem loop present in bacterial GlnRS. One designed GluRS variant is also highly specific for the tRNA(Gln2)(CUG) isoacceptor, which responds to the CAG codon, and shows no activity toward tRNA(Gln1)(UUG). Because it is now possible to eliminate particular codons from the genome of Escherichia coli, additional codons will become available for genetic code engineering. Isoacceptor-specific aminoacyl-tRNA synthetases will enable the reassignment of more open codons while preserving accurate encoding of the 20 canonical amino acids.

SUBMITTER: O'Donoghue P

PROVIDER: S-EPMC3251134 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase.

O'Donoghue Patrick P Sheppard Kelly K Nureki Osamu O Söll Dieter D

Proceedings of the National Academy of Sciences of the United States of America 20111207 51

The specificity of most aminoacyl-tRNA synthetases for an amino acid and cognate tRNA pair evolved before the divergence of the three domains of life. Glutaminyl-tRNA synthetase (GlnRS) evolved later and is derived from the archaeal-type nondiscriminating glutamyl-tRNA synthetase (GluRS), an enzyme with relaxed tRNA specificity capable of forming both Glu-tRNA(Glu) and Glu-tRNA(Gln). The archaea lack GlnRS and use a specialized amidotransferase to convert Glu-tRNA(Gln) to Gln-tRNA(Gln) needed fo ...[more]

PMID: 22158897

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Project description:Pseudomonas aeruginosa has a high potential for developing resistance to multiple antibiotics. The gene (glnS) encoding glutaminyl-tRNA synthetase (GlnRS) from P. aeruginosa was cloned and the resulting protein characterized. GlnRS was kinetically evaluated and the KM and kcat obs , governing interactions with tRNA, were 1.0 μM and 0.15 s-1 , respectively. The crystal structure of the α2 form of P. aeruginosa GlnRS was solved to 1.9 Å resolution. The amino acid sequence and structure of P. aeruginosa GlnRS were analyzed and compared to that of GlnRS from Escherichia coli. Amino acids that interact with ATP, glutamine, and tRNA are well conserved and structure overlays indicate that both GlnRS proteins conform to a similar three-dimensional structure. GlnRS was developed into a screening platform using scintillation proximity assay technology and used to screen ~2,000 chemical compounds. Three inhibitory compounds were identified and analyzed for enzymatic inhibition as well as minimum inhibitory concentrations against clinically relevant bacterial strains. Two of the compounds, BM02E04 and BM04H03, were selected for further studies. These compounds displayed broad-spectrum antibacterial activity and exhibited moderate inhibitory activity against mutant efflux deficient strains of P. aeruginosa and E. coli. Growth of wild-type strains was unaffected, indicating that efflux was likely responsible for the lack of sensitivity. The global mode of action was determined using time-kill kinetics. BM04H03 did not inhibit the growth of human cell cultures at any concentration and BM02E04 only inhibit cultures at the highest concentration tested (400 μg/ml). In conclusion, GlnRS from P. aeruginosa is shown to have a structure similar to that of E. coli GlnRS and two natural product compounds were identified as inhibitors of P. aeruginosa GlnRS with the potential for utility as lead candidates in antibacterial drug development in a time of increased antibiotic resistance.

Dataset Information

Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase.

Publications

Rational design of an evolutionary precursor of glutaminyl-tRNA synthetase.

Similar Datasets

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