Relationship of protein structure of isoleucyl-tRNA synthetase with pseudomonic acid resistance of Escherichia coli. A proposed mode of action of pseudomonic acid as an inhibitor of isoleucyl-tRNA synthetase.

To elucidate the mode of action of pseudomonic acid, we have compared the deduced amino acid sequences of isoleucyl-tRNA synthetases (ILeRS) from wild-type Escherichia coli strain MC4100, a pseudomonic acid-resistant mutant (strain PS102) of MC4100, and a pseudomonic acid-producing strain, Pseudomonas fluorescens. Compared with the wild-type enzyme, the deduced amino acid sequence of E. coli mutant ileS gene in strain PS102 shows a single amino acid substitution of leucine for phenylalanine at residue 594 of the IleRS. This mutational alteration in IleRS of an E. coli pseudomonic acid-resistant mutant resides in a region of the enzyme in close proximity to one of the consensus sequences of class I aminoacyl-tRNA synthetases, the KMSKS sequence between residues 602 and 606 of the E. coli IleRS. DNA sequence of the cloned ileS gene predicts that the P. fluorescens IleRS consists of 943 amino acids with 54% identity with the E. coli IleRS. The P. fluorescens ileS gene and the wild type and PS102 alleles of E. coli ileS were cloned into an expression vector, pEXPCR, and the sensitivities of E. coli DH5 alpha cells harboring each of these plasmids were compared. The cells harboring the P. fluorescens ileS were found to be most resistant to pseudomonic acid, while the transformants expressing the PS102 IleRS were more resistant than those containing the wild-type E. coli IleRS. IleRS purified from the wild-type E. coli was specifically cleaved by trypsin between Lys605 and Ser606 in the region of K602MSKS606. The protection of the IleRS from the trypsin digestion was found with pseudomonic acid or ATP, but not with isoleucine or tRNA(1Ile). Based on these results, we propose that pseudomonic acid binds to IleRS in the vicinity of the KMSKS sequence that is an ATP-binding subsite, and that pseudomonic acid is a bifunctional inhibitor with characteristics of both isoleucine and ATP, for example, an analog of isoleucyladenylate.