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Development of Methionyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Author(s) -
Robles Sara,
Hu Yanmei,
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.1066.4
Subject(s) - aminoacylation , aminoacyl trna synthetase , transfer rna , pseudomonas aeruginosa , enzyme , amino acid , biochemistry , methionine , biology , biosynthesis , chemistry , microbiology and biotechnology , rna , bacteria , gene , genetics
Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the enzymatic characterization and development of a screening platform based on methionyl tRNA synthetase (MetRS) from P. aeruginosa . Results MetRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases. The kinetic parameters for the interaction of P. aeruginosa MetRS with its three substrates (tRNA, ATP, and methionine) were determined. Initial velocities were determined for charging of tRNA using tRNA Met concentrations between 0.5 and 5 μM. The K M and V max was determined to be 29 μM and 3.0 μM min −1 , respectively, resulting in an observed k cat of 1.9 sec −1 . This resulted in a k cat / K M value of 0.067 s −1 μM −1 . The ATP:PP i exchange reaction was used to monitor interaction with ATP and methionine (Met). The K M , V max , observed k cat , and k cat / K M for interaction with ATP and Met was 515, 145, 24.2, 0.05 and 35.5, 223, 37.2, 1.05, all respectively. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa MetRS in a high throughput format. Using this assay, a natural product (800 compounds) and a synthetic compound (890 compounds) library was screened. Two compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM01F04 and BM01C11 inhibit the activity of P. aeruginosa MetRS with IC 50 values of 26 and 70 μM, respectively and both of these compounds exhibited promising MICs against Gram+ bacteria and mutant forms of Gram‐ bacteria. Conclusion MetRS 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) and the Howard Hughes Medical Institute (HHMI) Precollege and Undergraduate Science Education Program grant #52007568.