Peptidoglycan (PGN) constitutes the cell walls of virtually all bacteria, making it a target of the innate immune system.1,2 PGN is a polymer of alternating β (1→ 4) linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), crossbridged by oligopeptide stems.3–5 Lysostaphin type enzymes are believed to cleave the glycyl-glycine and glycyl-alanine bonds that occur in glycine-rich cross-bridges.6,7 Lysostaphins represent potential anti staphylococcal agents. Specifically, they can eradicate S. aureus nasal colonization in the rat model and are effective in treating methicillin-resistant S. aureus endophthalmitis in rabbits.8–10 These enzymes belong to metalloendopeptidase family and possess a conserved HXH active site motif. Together, D-Ala-D-Ala peptidase, the sonic hedgehog enzymes, the MepA-like enzymes, and the lysostaphine enzymes comprise the LAS enzyme superfamily.11 Unlike other LAS enzymes the lyostaphins are synthesized as proenzymes and require proteolytic processing for activation.12 To date, the only three-dimensional structure reported from the Lysostaphin type enzyme family is LytM from Staphylococcus aureus. Structures of both full length (residues 45–316; PDB ID: 1QWY) and truncated (residues 185–316; PDB ID: 2B44, 2BOP and 2B13) forms of LytM have been determined.12,13 Cleavage of LytM between residues 45 and 185 removes Asn317, which blocks substrate access to the active site in the longer, inactive form of the enzyme.12,13 The precise role(s) played by the lysostaphins in gram-negative bacteria remains unclear. In some gram-positive bacteria they participate in cell wall biogenesis.11 Horizontal transfer of genes responsible for cell-wall polysaccharide synthesis is correlated with virulence of some strains of Vibrio cholerae, such as O139 Bengal,14 suggesting that the cell-wall itself may represent a virulence factor. It is remarkable that Vibrio cholerae exerts its pathologic effects via a protein toxin on epithelial cells lining the small intestine that contains a modified form of GlcNAc.15
To better understand V. cholerae lysostaphin, the New York SGX Research Center for Structural Genomics (NYSGXRC; www.nysgxrc.org) targeted this protein (designated P01) for structure determination under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (PSI). We report the X-ray crystal structure of P01 determined at 1.9-Å resolution. Comparison with extant structures of LytM from S. aureus permitted identification of the catalytic domain of P01 and elucidation of its mechanisms of activation and catalysis.