Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase

Abstract

Sodium pseudomonate was shown to be a powerful competitive inhibitor of Escherichia coli B isoleucyl-tRNA synthetase (Ile-tRNA synthetase). The antibiotic competitively inhibits (Ki 6 nM; cf. Km 6.3 microM), with respect top isoleucine, the formation of the enzyme . Ile approximately AMP complex as measured by the pyrophosphate-exchange reaction, and has no effect on the transfer of [14C]isoleucine from the enzyme . [14C]Ile approximately AMP complex to tRNAIle. The inhibitory constant for the pyrophosphate-exchange reaction was of the same order as that determined for the inhibition of the overall aminoacylation reaction (Ki 2.5 nM; cf. Km 11.1 microM). Sodium [9'-3H]pseudomonate forms a stable complex with Ile-tRNA synthetase. Gel-filtration and gel-electrophoresis studies showed that the antibiotic is only fully released from the complex by 5 M-urea treatment or boiling in 0.1% sodium dodecyl sulphate. The molar binding ratio of sodium [9'-3H]pseudomonate to Ile-tRNA synthetase was found to be 0.85:1 by equilibrium dialysis. Aminoacylation of yeast tRNAIle by rat liver Ile-tRNA synthetase was also competitively inhibited with respect to isoleucine, Ki 20 microM (cf. Km 5.4 microM). The Km values for the rat liver and E. coli B enzymes were of the same order, but the Ki for the rat liver enzyme was 8000 times the Ki for the E. coli B enzyme. This presumably explains the low toxicity of the antibiotic in mammals.

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

“…Our determined K i value was 0.8 nM (Fig. 5B), which is in close agreement with the previously reported value of 2.5 nM for mupirocin with Ec-IleRS [39]. The calculated concentration of active Ec-IleRS in the assay was 33 nM.…”

“…Previous work on mupirocin revealed a K i of 2.5 nM for inhibition of tRNA aminoacylation by Ec-IleRS [39]. To determine the K i of this compound using the PPase/PNPase-coupled reaction, Ec-IleRS was tested in the absence of other aaRSs.…”

Development and validation of a liquid chromatographic method for in vitro mupirocin quantification in both skin layers and percutaneous penetration studies

“…Mupirocin was found to inhibit bacterial isoleucyl-tRNA synthetase [3,4], blocking protein synthesis and indirectly inhibiting RNA synthesis [5]. The last part of the epoxy chain presents a structural analogy with isoleucyn and it interacts reversible and specifically with the aminoacid at the active site of the enzyme.…”

“…The skin samples were clamped between the two chambers of Franz-diffusion cells, with the stratum corneum facing the donor compartment and the dermis facing the receptor one. The mupirocin solution was placed in the donor compartment and 0.4 7 ml samples were taken from the receptor side at different times (4,6,8,22 and 24 h) and replaced by the same volume of PBS. Samples were filtered and analyzed immediately.…”

How does pseudomonas fluorescens, the producing organism of the antibiotic pseudomonic acid A, avoid suicide?

“…isoleucyl adenylate complex, as measured by the pyrophosphate exchange reaction (Ki 6 X low9 M; cf. Km 6.3 X 10" M; pH 7.4,37'C) [3]. The binding ratio of sodium pseudomonate A to IRS is -1: 1.…”