Self-immunity guided identification of threonyl-tRNA synthetase as the molecular target of obafluorin, a β-lactone antibiotic

To meet the ever-growing demand of antibiotic discovery, new chemical matter and antibiotic targets are urgently needed. Many potent natural product antibiotics which were previously discarded can also provide lead molecules and drug targets. One such example is the structurally unique beta-lactone obafluorin, produced by Pseudomonas fluorescens ATCC 39502. Obafluorin is active against both Gram-positive and -negative pathogens, however the biological target was unknown. We now report that obafluorin targets threonyl-tRNA-synthetase and we identify a homologue, ObaO, which confers self-immunity to the obafluorin producer. Disruption of obaO in P. fluorescens ATCC 39502 results in obafluorin sensitivity, whereas expression in sensitive E. coli strains confers resistance. Enzyme assays demonstrate that E. coli threonyl-tRNA synthetase is fully inhibited by obafluorin, whereas ObaO is only partly susceptible, exhibiting a very unusual partial inhibition mechanism. Altogether, our data highlight the utility of a self-immunity guided approach for the identification of an antibiotic target de novo and will ultimately enable the generation of improved obafluorin variants.