Vancomycin does not affect the enzymatic activities of purified VanS(A)

VanS is a membrane-bound sensor histidine kinase responsible for sensing vancomycin and activating transcription of vancomycin-resistance genes. In the presence of vancomycin, VanS phosphorylates the transcription factor VanR, converting it to its transcriptionally active form. In the absence of vancomycin, VanS dephosphorylates VanR, thereby maintaining it in a transcriptionally inactive state. To date, the mechanistic details of how vancomycin modulates VanS activity have remained elusive. We have therefore studied these details in an in vitro system, using the full-length VanS and VanR proteins responsible for type-A vancomycin resistance in enterococci. Both detergent- and amphipol-solubilized VanS(A) display all the enzymatic activities expected for a sensor histidine kinase, with amphipol reconstitution providing a marked boost in overall activity relative to detergent solubilization. A putative constitutively activated VanS(A) mutant (T168K) was constructed and purified, and was found to exhibit the expected reduction in phosphatase activity, providing confidence that detergent-solubilized VanS(A) behaves in a physiologically relevant manner. In both detergent and amphipol solutions, VanS(A)’s enzymatic activities were found to be insensitive to vancomycin, even at levels many times higher than the antibiotic’s minimum inhibitory concentration. This result argues against direct activation of VanS(A) via formation of a binary antibiotic-kinase complex, suggesting instead that either additional factors are required to form a functional signaling complex, or that activation does not require direct interaction with the antibiotic.