| Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme which attaches tryptophan to tRNATrp to provide tryptophanyl-tRNA for protein synthesis. Indolmycin, an inhibitor of bacterial growth, is a tryptophan analogue that competes with the cognate amino acid for TrpRS. To investigate the interaction of indolmycin with TrpRS, random mutagenesis of the Bacillus stearothermophilus trpS gene was performed, followed by transformation of Escherichia coli with the trpS mutants and subsequent growth of the transformants in the presence of indolmycin. Colonies obtained in this fashion were re-screened for indolmycin resistance. 209 clones were picked and their trpS sequence determined: 18 contained single base substitutions in 10 different positions in the trpS gene, and the remaining clones contained 2–8 point mutations. Analysis of the crystal structure of B. stearothermophilus complexed with ATP and indolmycin revealed that all single point mutations are >20Å from the active site and located in the putative tRNATrp, binding site. Steady-state kinetic analysis of five selected mutant TrpRS enzymes revealed that each had variable effects on KM, kcat, kcat/KM, and Ki of tryptophan, tRNA, ATP, and indolmycin substrates, by an average change of 2-fold. The moderate changes in kinetic parameters of these mutants are sufficient to cause indolmycin resistance. Since our mutant selection protocol required substantial growth on indolmycin plates, we might have precluded the isolation of active site trpS mutants as their corresponding enzymes would probably have been damaged. In order to shed more light on the structural changes making an indolmycin resistant enzyme, the crystal structure of mutant Y265F TrpRS complexed with ATP and indolmycin was determined at 1.95Å resolution. Compared to the wild-type TrpRS structure the mutant displayed a 90° rotation of the imidazole ring of H43, an active site residue involved in indolmycin binding.; To clarify the selectivity of indolmycin for prokaryotic TrpRS enzymes, the inhibitory properties of indolmycin were determined for human TrpRS. This eukaryotic enzyme was highly refractive to indolmycin inhibition even under the highest inhibitor conditions experimentally possible. These data explain the earlier suggestion that indolmycin is a selective inhibitor for prokaryotic TrpRS enzymes (Werner et al., 1976). |
