Characterization and structure determination of prolyl‐tRNA synthetase from <i>Pseudomonas aeruginosa</i> and development as a screening platform

Pena, Noah; Dranow, David M.; Hu, Yanmei; Escamilla, Yaritza; Bullard, James M.

doi:10.1002/pro.3579

Cited by 7 publications

(8 citation statements)

References 26 publications

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“…Its impact is particularly pronounced in Plasmodium falciparum cytoplasmic ProRS, an E‐type enzyme, where it concurrently binds to the L‐Pro and tRNA Pro sites with a half‐maximal inhibitory concentration (IC 50 ) value of approximately 10 nM (Jain et al, 2015). However, HF exhibits significantly reduced efficacy, approximately 100‐fold less, against the P‐type ProRS, exemplified by Pseudomonas aeruginosa ProRS (Pena et al, 2019). This observation prompted us to investigate whether TtProRS, classified as an E‐type enzyme, retained a heightened sensitivity to HF.…”

Section: Resultsmentioning

confidence: 99%

“…The IC 50 values of HF for P. falciparum , T. gondii , and human ProRSs are 9.0, 5.41, and 10.65 nM, respectively (Jain et al, 2015; Manickam et al, 2022). However, HF is much less effective (~100‐fold) against the P‐type ProRS (such as Pseudomonas aeruginosa ProRS) (Pena et al, 2019). Despite TtProRS possessing a sequence resembling that of an E‐type ProRS, it had an IC 50 value of HF ~24‐fold higher than that for the typical E‐type ProRS.…”

Section: Discussionmentioning

confidence: 99%

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Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate

Latifah,

Ivanesthi,

Tseng

et al. 2024

Protein Science

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Prolyl‐tRNA synthetase (ProRS), belonging to the family of aminoacyl‐tRNA synthetases responsible for pairing specific amino acids with their respective tRNAs, is categorized into two distinct types: the eukaryote/archaeon‐like type (E‐type) and the prokaryote‐like type (P‐type). Notably, these types are specific to their corresponding cognate tRNAs. In an intriguing paradox, Thermus thermophilus ProRS (TtProRS) aligns with the E‐type ProRS but selectively charges the P‐type tRNAPro, featuring the bacterium‐specific acceptor‐stem elements G72 and A73. This investigation reveals TtProRS's notable resilience to the inhibitor halofuginone, a synthetic derivative of febrifugine emulating Pro‐A76, resembling the characteristics of the P‐type ProRS. Furthermore, akin to the P‐type ProRS, TtProRS identifies its cognate tRNA through recognition of the acceptor‐stem elements G72/A73, along with the anticodon elements G35/G36. However, in contrast to the P‐type ProRS, which relies on a strictly conserved R residue within the bacterium‐like motif 2 loop for recognizing G72/A73, TtProRS achieves this through a non‐conserved sequence, RTR, within the otherwise non‐interacting eukaryote‐like motif 2 loop. This investigation sheds light on the adaptive capacity of a typically conserved housekeeping enzyme to accommodate a novel substrate.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate

Latifah,

Ivanesthi,

Tseng

et al. 2024

Protein Science

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“…Prolyl-tRNA synthetase belongs to the ubiquitously expressed enzyme family of aminoacyl-tRNA synthetases (aaRS), performing a crucial role in protein biosynthesis, cell growth, and viability. Enzyme of bacterial origin is different from the eukaryotic one and widely studied as a promising antimicrobial drug target [59]. Second strongest interaction with aerolysin was that of M4 (MEROPS) protease of the zinc metallopeptidase family.…”

Section: Discussionmentioning

confidence: 99%

Co-Prevalence of Virulence and Pathogenic Potential in Multiple Antibiotic Resistant Aeromonas spp. from Diseased Fishes with In Silico Insight on the Virulent Protein Network

Chakraborty

Das

Bera

et al. 2022

Life

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Aeromonas species exhibit widespread presence in food, poultry, and aquaculture. They are major multi-drug-resistant fish pathogens. This study aims to identify Aeromonas species harbouring virulence genes aerolysin, flagellin, and lipase from diseased fishes of Assam wetlands with association with antibiotic resistance and in vivo pathogenicity. One hundred and thirty-four Aeromonas strains were isolated and thirty representative species identified using genus-specific 16S rRNA gene amplification. A. veronii was most prevalent (53.7%) followed by A. hydrophila (40.2%), A. caviae (4.47%), and A. dhakensis (1.49%). Ninety percent (90%) of strains harboured at least one of the studied virulence genes: aerA (73.3%), lip (46.6%), and flaA (26.6%). The highest multiple antibiotic resistance (MAR) index 0.8 corresponded to A. hydrophila DBTNE1 (MZ723069), containing all the studied genes. The lowest LD50 values (1.6 × 106 CFU/fish) corresponded to isolates having both aerA and lip. β-lactams showed utmost resistance and lowest for aminoglycosides. There was a significant (p < 0.05) Pearson chi-square test of association between the occurrence of virulence and antibiotic resistance. The in silico protein–protein interaction revealed important drug targets, such as σ28 transcription factor, aminoacyl-tRNA synthetase, and diacylglycerol kinase, with significant (p < 0.05) enrichment. This study suggests that fish-isolate Aeromonas strains represent potential threat to aquaculture with subsequent risk of transferring antibiotic resistance to human pathogens.

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“…Timed aminoacylation reactions were measured using SPA technology as described. 12 Briefly, reactions (50 µL) were carried out in 96-well plates (costar). A master mix (35 μL) contained components to result in final concentrations of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 2.5 mM ATP, 0.5 mM spermine, 75 µM [ 3 H] Tyr, 1 mM DTT, and either 0.0015 µM PaTyrRS-Z or 0.05 µM PaTyrRS-S.…”

Section: Methodsmentioning

confidence: 99%

“… Authors’ Note The authors disclose that portions of the Materials and Methods section were previously described in the following articles and are described here for clarity and descriptive purposes: Balboa et al, 24 Hu et al, 11 Pena et al, 12 Escamilla et al (Glutaminyl-tRNA Synthetase from Pseudomonas aeruginosa : Characterization, Structure, and Development as a Screening Platform. Protein Sci.…”

mentioning

confidence: 99%

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

et al. 2020

Self Cite

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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, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition.

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

Cited by 7 publications

References 26 publications

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate

Adaptive evolution: Eukaryotic enzyme's specificity shift to a bacterial substrate

Co-Prevalence of Virulence and Pathogenic Potential in Multiple Antibiotic Resistant Aeromonas spp. from Diseased Fishes with In Silico Insight on the Virulent Protein Network

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds

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