Probing the mechanism of peptidoglycan amidase activation by FtsEX-EnvC.

The FtsEX-EnvC-AmiA/B system is a key component of the cell division machinery that directs breakage of the peptidoglycan layer during separation of daughter cells. Structural and mechanistic studies have shown that ATP binding by FtsEX in the cytoplasm drives periplasmic conformational changes in EnvC, which lead to the binding and activation of peptidoglycan amidases such as AmiA and AmiB. The FtsEX-EnvC amidase system is highly regulated to prevent cell lysis with at least two separate layers of autoinhibition that must be relieved to initiate peptidoglycan hydrolysis during division. Here, we test the FtsEX-EnvC amidase activation mechanism through site-directed mutagenesis. We identify mutations that disrupt the autoinhibition mechanism of FtsEX-EnvC and an N-terminal deletion variant that prevents activation. Finally, we develop a cysteine locking residue pair that stabilizes the complex in its amidase activating conformation. The reported EnvC variants greatly enhance our understanding of the FtsEX-EnvC autoinhibition mechanism and the conformational changes underpinning amidase activation. Our observations are consistent with the proposed mechanism of amidase activation by large-scale conformational changes in FtsEX-EnvC, allowing recruitment and activation of peptidoglycan amidases.IMPORTANCEIn , the FtsEX-EnvC system regulates two of the three division-associated amidases that break the peptidoglycan layer during bacterial division. Structural and mechanistic studies have revealed a detailed molecular mechanism for amidase activation in which an ABC transporter and its periplasmic partner reversibly activate periplasmic amidases under direction of the cytoplasmic cell division machinery. This paper explores structural features of EnvC that underpin autoinhibition and the activation mechanism. The FtsEX-EnvC system serves as a powerful example of a Type VII ABC transporter that uses transmembrane conformational changes to drive work in the periplasmic space.