Lysyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Identification of Inhibitory Compounds

Abstract

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 K_m values for the interaction with lysine, adenosine triphosphate (ATP), and tRNA^Lys were determined to be 45.5, 627, and 3.3 µM, respectively. The k_cat^obs values were calculated to be 13, 22.8, and 0.35 s^-1, resulting in k_cat^obs/K_M values of 0.29, 0.036, and 0.11 s^-1µM^-1, respectively. Using scintillation proximity assay technology, natural product and synthetic compound libraries were screened to identify inhibitors of function of the enzyme. Three compounds (BM01D09, BT06F11, and BT08F04) were identified with inhibitory activity against LysRS. The IC₅₀ values were 17, 30, and 27 µM for each compound, respectively. The minimum inhibitory concentrations were determined against a panel of clinically important pathogens. All three compounds were observed to inhibit the growth of gram-positive organisms with a bacteriostatic mode of action. However, two compounds (BT06F11 and BT08F04) were bactericidal against cultures of gram-negative bacteria. When tested against human cell cultures, BT06F11 was not toxic at any concentration tested, and BM01D09 was toxic only at elevated levels. However, BT08F04 displayed a CC₅₀ of 61 µg/mL. In studies of the mechanism of inhibition, BM01D09 inhibited LysRS activity by competing with ATP for binding, and BT08F04 was competitive with ATP and uncompetitive with the amino acid. BT06F11 inhibited LysRS activity by a mechanism other than substrate competition.

Keywords: Pseudomonas aeruginosa; aminoacyl-tRNA synthetase; antibiotics; drug discovery; lysyl-tRNA synthetase (LysRS); protein synthesis.

Figure 1.

Alignment of the amino acid sequence of P. aeruginosa lysyl-tRNA synthetase (LysRS) with homologs. The protein sequences of LysRS from Bt, Burkholderia thailandensis; Ec, E. coli; Pa, P. aeruginosa; and Tth, T. thermophilus were downloaded from the National Center for Biotechnology Information. Accession numbers for LysRS protein sequences of B. thailandensis, E. coli (S), E. coli (U), P. aeruginosa, and T. thermophilus are AOJ57238, AAA83071, Q8FAT5, NP_252390, and P41255, respectively. Sequence alignments were performed using Vector NTI Advance 11.5.4 (Invitrogen). Identical residues are indicated by white letters on a black background, whereas similar sequences are black letters on a gray background. The three structural motifs (1, 2, and 3) are indicated. Amino acids that interact with adenosine triphosphate (●), lysine (◼), and tRNA^Lys (◆) are indicated above the aligned residues.

Figure 2.

Determination of the activity of P. aeruginosa lysyl-tRNA synthetase (LysRS) and the kinetic parameters governing interactions with its three substrates: adenosine triphosphate (ATP), lysine and tRNA^Lys. P. aeruginosa LysRS was titrated into the aminoacylation assay (A) as described in the “Methods and Materials” section in amounts varying from 0.5 to 4 pmol enzyme. Background activity was minimal and was subtracted from values at all concentrations of LysRS. Initial velocities for the interaction of LysRS with both ATP (B) and lysine (C) were determined using the ATP:PP_i exchange reaction. The initial velocity for the interaction of LysRS with tRNA was determined using the aminoacylation reaction (D). The concentration of LysRS in the aminoacylation reactions and the exchange reactions was 0.03 μM and 0.1 μM, respectively. Initial velocities were determined, and the data were fit to a Michaelis-Menten steady-state model using XLfit 5.3 (IDBS) to determine K_M and V_max. The kinetic parameters were calculated from these data for the interaction of LysRS with the three substrates (E).

Figure 3.

The chemical structure of the hit compounds. The structure of BM01D09 (A), BT06F11 (B), and BT08F04 (C).

Figure 4.

Characterization of enzymatic and bacterial inhibition by BM01D09, BT06F11 and BT08F04. IC₅₀ values for the inhibitory potency of BM01D09 (A), BT06F11 (B), and BT08F04 (C) against the aminoacylation activity of P. aeruginosa LysRS were 17 μM, 30 μM, and 27 μM, respectively. The compounds were serially diluted from 200 μM to 0.4 μM into aminoacylation assays containing P. aeruginosa LysRS at 0.01 μM. “% Positive” indicates the percentage of activity observed relative to activity in assays in which only DMSO was added to the assay in the absence of compound. The curve fits and IC₅₀ values were determined using the sigmoidal dose-response model in XLfit 5.3 (IDBS). The activity of the hit compounds against the growth of cultures containing (D) S. aureus and (E) M. catarrhalis bacteria were determined using broth microdilution susceptibility testing. Compounds were added to bacterial cultures at 4×minimum inhibitory concentration. Samples were analyzed by plating and determination of colony-forming units at 0, 2, 4, 6, and 24 h. Open circles (○) represent cultures containing BM01D09, filled squares (◼) represent cultures containing BT06F11, and filled triangles (▲) represent cultures containing BT08F04. Filled circles (●) represent the growth of control cultures containing only DMSO in the absence of compound.

Figure 5.

Hit compound competition with adenosine triphosphate (ATP) and lysine for active site binding. In aminoacylation assays containing BM01D09, the IC₅₀ values increased as the concentration of ATP was increased, indicating competitive inhibition (A). In competition assays containing BT08F04, the IC₅₀ values increased with increasing concentrations of ATP, indicating competitive inhibition (B), and decreased when the concentration of lysine increased, which is characteristic of an uncompetitive inhibitor (C). P. aeruginosa lysyl-tRNA synthetase (LysRS) concentration was set at 0.01 μM in these assays. Background amounts of free [³H]Lys in the absence of LysRS were insignificant.

Figure 6.

Toxicity of the hit compounds in cultures of human cell lines. Toxicity of the hit compounds was measured using human embryonic kidney 293 (HEK-293) cell cultures. The compound concentration ranged from 25 to 400 μg/mL. The data points represent the average values for assays carried out in triplicate. “% Positive” indicates the percentage of activity observed relative to activity in assays in which only DMSO was added to the assay in the absence of compound. The curve fits and CC₅₀ values were determined using the sigmoidal dose-response model in XLfit 5.3 (IDBS). (A) BT06F11 was not toxic at any concentration tested. The hatched column represent the growth of cultures in the positive control assays, and the solid column represents the cultures containing the indicated concentration of BT06F11. The CC₅₀ values for (B) BM01D09 and (C) BT08F04 were 370 μg/mL and 61 μg/mL, respectively. The control staurosporine (D) was serially diluted in assays from 1 to 0.001 μg/mL and exhibited a CC₅₀ of 0.003 μg/mL.