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Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

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Published on: June 30, 2016

Characterization and structure determination of prolyl-tRNA synthetase from Pseudomonas aeruginosa and development as

Noah Pena¹, David M Dranow^2,3, Yanmei Hu¹

¹The University of Texas - RGV, Edinburg, Texas 78541.

Protein Science : a Publication of the Protein Society

|January 23, 2019

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 K_M values for interactions with ATP, proline, and tRNA were 154, 122, and 5.5 μM, respectively. The turn-over numbers, k_cat^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 α₂ 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.

Insights

This study characterizes prolyl-tRNA synthetase (ProRS) from Pseudomonas aeruginosa, a multi-drug resistant pathogen. A screening platform was developed, identifying two inhibitory compounds against this essential bacterial enzyme.

Area of Science:

Microbiology
Structural Biology
Biochemistry

Background:

Pseudomonas aeruginosa is an opportunistic, multi-drug resistant pathogen causing significant infections.
Prolyl-tRNA synthetase (ProRS) is essential for bacterial protein synthesis and a potential drug target.

Purpose of the Study:

To characterize the enzyme prolyl-tRNA synthetase (ProRS) from Pseudomonas aeruginosa.
To develop and validate a screening platform for identifying inhibitors of P. aeruginosa ProRS.

Main Methods:

Overexpression and purification of P. aeruginosa ProRS in E. coli.
Kinetic evaluation and X-ray crystallography of P. aeruginosa ProRS.
Development of a scintillation proximity assay (SPA) for high-throughput screening.

Main Results:

Kinetic parameters (K_M, k_cat^obs) for ProRS with ATP, proline, and tRNA were determined.
The crystal structure of P. aeruginosa ProRS was solved at 2.60 Å resolution, revealing conserved active site residues.
Screening of 890 compounds identified two inhibitors, BT06A02 and BT07H05.

Conclusions:

The characterized P. aeruginosa ProRS exhibits conserved structural and functional properties.
A robust SPA-based screening platform utilizing ProRS was successfully established.
This work validates ProRS as a viable target for antimicrobial drug discovery.

Read full analysis

Keywords:

Pseudomonas aeruginosa aminoacyl-tRNA synthetase antibiotics drug discovery protein synthesis proyl-tRNA synthetase

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