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Development of Arginyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Author(s) -
Cantu Daniel,
Salazar Humberto,
Bullard James M
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1066.5
Subject(s) - pseudomonas aeruginosa , aminoacyl trna synthetase , aminoacylation , enzyme , biochemistry , bacteria , transfer rna , escherichia coli , amino acid , biosynthesis , chemistry , biology , microbiology and biotechnology , rna , gene , genetics
Pseudomonas aeruginosa is a common cause of nosocomial infections and the leading cause of mortality in patents with cystic fibrosis. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. Results P. aeruginosa arginyl‐tRNA synthetase (ArgRS) was overexpressed in Escherichia coli cells, and purified to homogeneity. ArgRS from P. aeruginosa is a discriminating synthetase with respect to the requirement for the presence of tRNA Arg to produce a stable arginyl‐AMP intermediate. The kinetic parameters for interaction with tRNA were determined and the K M and V max were 4.0 μM and 11.7 μM min −1 , respectively. The observed k cat was calculated to be 0.32 s −1 resulting in a k cat / K M value of 0.08 s −1 μM −1 . Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa ArgRS in a high throughput format. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with the ability to inhibit function of the enzyme. Four compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM06E07, BT04F10, BT10B04 and BT11F03 inhibit the activity of P. aeruginosa ArgRS with IC 50 values of 29.2, 35.4, 31.6 and 8.1 μM, respectively. All four of these compounds exhibited promising MICs against Gram+ bacteria and moderate activity against Gram‐ bacteria causing respiratory infections. Conclusion ArgRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms. Support or Funding Information Research was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017).