Characterization of VldE (Spr1875), a Pneumococcal\nTwo-State l,d‑Endopeptidase with a Four-Zinc\nCluster in the Active Site

Remodeling of the\npneumococcal cell wall, carried out by peptidoglycan\n(PG) hydrolases, is imperative for maintaining bacterial cell shape\nand ensuring survival, particularly during cell division or stress\nresponse. The Streptococcus pneumoniae protein Spr1875 plays a role in stress response, both regulated\nby the VicRK two-component system (analogous to the WalRK TCS found\nin Firmicutes). Modular Spr1875 presents a putative cell-wall binding\nmodule at the N-terminus and a catalytic C-terminal module (Spr1875<sup>MT3</sup>) connected by a long linker. Assays of the full-length\nprotein and Spr1875<sup>MT3</sup> with PG-based synthetic substrates\nby liquid chromatography/mass spectrometry revealed Spr1875 as an l,d-endopeptidase, renamed VldE (for VicRK-regulated l,d-endopeptidase), which hydrolyzed the cross-linked\nstem peptide in the PG. Remarkably, we observed asymmetric turnover\nwith specific recognition of the acceptor peptide strand. Localization\nexperiments showed that the protein is directed to the septum, which\nsuggests that muralytic activity could be required for pneumococcal\ngrowth under stress conditions. Our findings, based on six high-resolution\nX-ray crystallographic structures and molecular-dynamics simulations,\nreveal two states for VldE<sup>MT3</sup>. The protein transitions\nbetween a noncatalytic state that binds up to four zinc ions, thus\nbehaving as a Zn<sup>2+</sup> reservoir, and a catalytic state that\nperforms the hydrolytic reaction with a single zinc ion. Furthermore,\ncomputational studies provide insight into the mechanism of catalytic-water\nactivation and nucleophilic attack on the specific scissile peptide\nbond of the asymmetric cross-linked PG.