Chemistry Commons^™

Two Forms Of Tyrosyl-Trna Synthetase From Pseudomonas Aeruginosa: Characterization And Discovery Of Inhibitory Compounds, Casey Anne Hughes, Varesh Gorabi, Yaritza Escamilla, Frank B. Dean, James M. Bullard

Chemistry Faculty Publications and Presentations

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes (tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s–1 and 3.1, 3.8, and 1.9 s–1, …

Glutaminyl-Trna Synthetase From Pseudomonas Aeruginosa: Characterization, Structure, And Development As A Screening Platform, Yaritza Escamilla, Casey A. Hughes, Jan Abendroth, David M. Dranow, Samantha Balboa, Frank B. Dean, James M. Bullard

Chemistry Faculty Publications and Presentations

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 kcatobs , 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 …

Lysyl-Trna Synthetase From Pseudomonas Aeruginosa: Characterization And Identification Of Inhibitory Compounds, Samantha Balboa, Yanmei Hu, Frank B. Dean, James M. Bullard

Chemistry Faculty Publications and Presentations

Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial infections and has highly developed systems for acquiring resistance against numerous antibiotics. The gene (lysS) encoding P. aeruginosa lysyl-tRNA synthetase (LysRS) was cloned and overexpressed, and the resulting protein was purified to 98% homogeneity. LysRS was kinetically evaluated, and the Km values for the interaction with lysine, adenosine triphosphate (ATP), and tRNALys were determined to be 45.5, 627, and 3.3 µM, respectively. The kcatobs values were calculated to be 13, 22.8, and 0.35 s−1, resulting in kcatobs/KM values of 0.29, 0.036, and 0.11 s−1µM−1, …

Discovery And Characterization Of Chemical Compounds That Inhibit The Function Of Aspartyl-Trna Synthetase From Pseudomonas Aeruginosa, Araceli Corona, Stephanie O. Palmer, Regina Zamacona, Benjamin Mendez, Frank B. Dean, James M. Bullard

Chemistry Faculty Publications and Presentations

Pseudomonas aeruginosa, an opportunistic pathogen, is highly susceptible to developing resistance to multiple antibiotics. The gene encoding aspartyl-tRNA synthetase (AspRS) from P. aeruginosa was cloned and the resulting protein characterized. AspRS was kinetically evaluated, and the KM values for aspartic acid, ATP, and tRNA were 170, 495, and 0.5 μM, respectively. AspRS was developed into a screening platform using scintillation proximity assay (SPA) technology and used to screen 1690 chemical compounds, resulting in the identification of two inhibitory compounds, BT02A02 and BT02C05. The minimum inhibitory concentrations (MICs) were determined against nine clinically relevant bacterial strains, including efflux pump …

Identification Of Chemical Compounds That Inhibit The Function Of Histidyl-Trna Synthetase From Pseudomonas Aeruginosa, Yanmei Hu, Stephanie O. Palmer, Sara Robles, Tahyra Resto, Frank Dean, James M. Bullard

Chemistry Faculty Publications and Presentations

Pseudomonas aeruginosa histidyl-tRNA synthetase (HisRS) was selected as a target for antibiotic drug development. The HisRS protein was overexpressed in Escherichia coli and kinetically evaluated. The KM values for interaction of HisRS with its three substrates, histidine, ATP, and tRNAHis, were 37.6, 298.5, and 1.5 μM, while the turnover numbers were 8.32, 16.8, and 0.57 s-1, respectively. A robust screening assay was developed, and 800 natural products and 890 synthetic compounds were screened for inhibition of activity. Fifteen compounds with inhibitory activity were identified, and the minimum inhibitory concentration (MIC) was determined for each against a panel of nine pathogenic …

Identification And Characterization Of A Chemical Compound That Inhibits Methionyl-Trna Synthetase From Pseudomonas Aeruginosa, Sara Robles, Yanmei Hu, Tahyra Resto, Frank Dean, James M. Bullard

Chemistry Faculty Publications and Presentations

Background: Pseudomonas aeruginosa is an opportunistic pathogen problematic in causing nosocomial infections and is highly susceptible to development of resistance to multiple antibiotics. The gene encoding methionyl-tRNA synthetase (MetRS) from P. aeruginosa was cloned and the resulting protein characterized.

Methods: MetRS was kinetically evaluated and the KM for its three substrates, methionine, ATP and tRNAMet were determined to be 35, 515, and 29 μM, respectively. P. aeruginosaMetRS was used to screen two chemical compound libraries containing 1690 individual compounds.

Results: A natural product compound (BM01C11) was identified that inhibited the aminoacylation function. The compound inhibited P. aeruginosa MetRS with an …