The long chain fatty acid-CoA ligase VraA plays a regulatory role in vancomycin resistance in Staphylococcus aureus.

The pathogen Staphylococcus aureus threatens clinical and public health. Vancomycin, a former last-resort treatment, has waning efficacy against resistant strains (VISA) that alter their cell envelope using enzymes like VraA. However, the specific functional mechanism of VraA in VISA is still not understood. Here, we successfully obtained monomeric VraA and found that oleate is the optimal substrate for VraA. VraA was characterized as a peripheral membrane protein with a hydrodynamic radius of 5.7 ± 0.3 nm. Disruption of VraA significantly increased S. aureus susceptibility to vancomycin, as demonstrated by minimum inhibitory concentration (MIC) assays and RT-qPCR. The structural model of VraA predicted using AlphaFold2 was refined via ModRefiner, and active site residues were identified. Our findings underscore the pivotal role of VraA in mediating vancomycin resistance in S. aureus, providing valuable insights for developing targeted therapies against this pathogen.