Dataset Information

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform.

ABSTRACT: Pseudomonas aeruginosa is an opportunistic multi-drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl-tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. ProRS was kinetically evaluated and the KM values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 ?M, respectively. The turn-over numbers, kcat obs , for interactions with these substrates were calculated to be 5.5, 6.3, and 0.2 s-1 , respectively. The crystal structure of the ?2 form of P. aeruginosa ProRS was solved to 2.60?Å resolution. The amino acid sequence and X-ray crystal structure of P. aeruginosa ProRS was analyzed and compared with homologs in which the crystal structures have been solved. The amino acids that interact with ATP and proline are well conserved in the active site region and overlay of the crystal structure with ProRS homologs conforms to a similar overall three-dimensional structure. ProRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 890 chemical compounds, resulting in the identification of two inhibitory compounds, BT06A02 and BT07H05. This work confirms the utility of a screening system based on the functionality of ProRS from P. aeruginosa.

SUBMITTER: Pena N

PROVIDER: S-EPMC6423717 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform.

Pena Noah N Dranow David M DM Hu Yanmei Y Escamilla Yaritza Y Bullard James M JM

Protein science : a publication of the Protein Society 20190206 4

Pseudomonas aeruginosa is an opportunistic multi-drug resistant pathogen implicated as a causative agent in nosocomial and community acquired bacterial infections. The gene encoding prolyl-tRNA synthetase (ProRS) from P. aeruginosa was overexpressed in Escherichia coli and the resulting protein was characterized. ProRS was kinetically evaluated and the K<sub>M</sub> values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 μM, respectively. The turn-over numbers, k<sub>cat</sub> ...[more]

PMID: 30666738

Similar Datasets

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

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform.

Publications

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as a screening platform.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets