Peptidoglycan hydrolases represent an alternative to small molecule antibacterials, despite concerns relating to immunogenicity, the release of proinflammatory components during bacteriolysis and the development of resistance [3]. The peptidoglycan endopeptidases lysostaphin and LytM cleave the characteristic pentaglycine crossbridges of S. aureus peptidoglycan [4–6] and are therefore of interest as potential antistaphylococcal agents. Lysostaphin (Figure 1) is produced by Staphylococcus simulans biovar staphylolyticus. The secreted preproprotein is synthesized with a leader

sequence, proregion, catalytic domain, and the cell wall targeting domain (CWT) [7]. The low AC220 clinical trial complexity proregion consists of a variable number of stereotypical repeats (sequence [8]. It can be cleaved off in vivo

by extracellular cysteine protease [9] to release the mature form, which is often simply https://www.selleckchem.com/products/tubastatin-a.html H 89 called lysostaphin and is commercially available. Mature lysostaphin consists of the catalytic and CWT domains. The catalytic domain belongs to MEROPS family 23 in clan MO [10] and can be classified with the LAS metallopeptidases [11]. Sequence alignments suggest that the single Zn2+ ion in the active site is coordinated by His279, Asp283 and His362 (numbering according to Swiss-Prot entry P10547) and a water molecule. As the name implies, the CWT domain anchors the protein to cell walls [9] (Figure 1). Figure 1 Domain organization of preprolysostaphin and full-length LytM. (A) Schematic representation of the domain organization of preprolysostaphin and full-length LytM. The alignment shows the high similarity of the two proteins in the region of the catalytic domain. The Zn2+ ligands see more of the mature forms in the Hx3D and HxH motifs are highlighted in bold. Those in the Hx3D motif are separately changed to alanines in the mutationally inactivated LytM variants. (B) Schematic representation of lysostaphin, LytM, and the LytM fragments that are used for this study. (C) Overall (top)

and active site region (bottom) representations of the three-dimensional structures of preprolysostaphin (left) and full-length LytM (right). The left overall model was generated by the SWISSPROT server based on PDB entries 1QWY [12] and 1R77 [13]. The relative orientation of the catalytic and CWT domains is unknown and was chosen arbitrarily. It is not known whether the proregion repeats assume a defined structure or remain unstructured. The right overall model is an experimental structure directly based on PDB entry 1QWY [12]. The biological role of lysostaphin is well established. The (mature) protein is inactive against the producer organism, but very effective in cleaving S. aureus cell walls [14]. This property has made the enzyme attractive as an antibacterial agent [15–21].