Kinetic properties of pure overproduced Bacillus subtilis phenylalanyl‐tRNA synthetase do not favour its in vivo inhibition by ochratoxin A

Abstract

Ochratoxine A (OTA) inhibits growth of Bacillus subtilis at pHs below 7. Since OTA is a phenylalanine analogue, this effect could be due to inhibition of phenylalanine-tRNA synthetase (PheRS) by competition of this mycotoxin with the amino acid. Homogeneous PheRS was purified from Bacillus subtilis and from E. coli transformed with the PheRS gene. The latter produced about 40 times more PheRS than R subtilis. The K m and K, values of PheRS, respectively, for phenylalanine and OTA were measured and their concentrations within the cell determined. It appears that the concentration of OTA in the cell, in spite of a 25-fold accumulation, remained too low to significantly compete with phenylalanine. This does not suggest PheRS to be the target of OTA in cell growth and protein synthesis inhibition in Bacillus subtilis. It was also shown that the 2-3-fold increase of PheRS in OTA-treated cells is not due to phenylalanine-controUed attenuation regulation.

Ochratoxin Binding to Phenylalanyl-tRNA Synthetase: Computational Approach to the Mechanism of Ochratoxicosis and Its Antagonism

“…A hydrogen bond is formed between Arg204 and the amide carbonyl oxygen of OA, and the phenyl group leans against Met148 (Table ). This binding mode is marked by relatively few strong contacts between the ligand and the enzyme, which suggests weak binding, as observed experimentally by Roth et al and Creppy et al , …”

“…A hydrogen bond is formed between Arg204 and the amide carbonyl oxygen of OA, and the phenyl group leans against Met148 (Table 1). This binding mode is marked by relatively few strong contacts between the ligand and the enzyme, which suggests weak binding, as observed experimentally by Roth et al and Creppy et al 12,13 Additional sites may exist on the periphery of the tetrameric enzyme where OA could weakly bind, analogously to this site. The virtue of this site is that it could explain the antagonistic action of aspartame on OA toxicity.…”

“…In 1993, Roth and co-workers repeated the experiments of Konrad and Ro ¨schenthaler using specially purified PheRSBS and obtained values of K m Phe ) 2.8 × 10 -5 M and K i Phe ) 4.33 × 10 -3 M. 13 These data, viewed alone, would seem to support the in vivo inhibition of PheRS by OA more so than the results of Creppy et al However, Roth et al's measurement of the intracellular concentrations of OA and Phe, while demonstrating a 20-fold enrichment of OA, revealed concentrations of [Phe] ) 3 × 10 -4 M and [OA] ) 1 × 10 -3 M. From these data, the percent inhibition is calculated to be a mere 2%. 12 Thus, Roth et al dismiss the hypothesis that competitive inhibition of PheRS by OA is responsible for the latter's toxicity.…”

“…16 Its major mechanism of action was believed to be prevention of ochratoxin-induced inhibition of protein synthesis, although it was unclear whether this antagonism should be ascribed to aspartame itself or to Phe produced by the metabolism of aspartame, which yields Phe, aspartic acid, and methanol. The recent suggestions that PheRS may not be involved in ochratoxicosis, 13,14 however, indicate that aspartame may have a different mode of antagonism. Indeed, Baudrimont et al found that aspartame inhibits the binding of OA to plasma proteins, an effect also observed with the nonsteroidal antiinflammatory drug piroxicam.…”

New Class of Bacterial Phenylalanyl-tRNA Synthetase Inhibitors with High Potency and Broad-Spectrum Activity

“…For ochratoxin, the food-contaminating mycotoxin, inhibition of Phe-RS was initially discussed as a possible mechanism of toxicity (23). Subsequent investigators question this interpretation, nevertheless, as the concentration of ochratoxin in Bacillus subtilis appears to be too low to significantly compete with phenylalanine for the binding site of Phe-RS (33). For other aa-RS enzymes several inhibitors have been patented and reported in the literature over the years (6,16,40), but none of them has been developed as an antibacterial agent so far.…”