Conserved loop residues-Tyr and Asn near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis.

Lysostaphin endopeptidase cleaves pentaglycine cross-bridges found in staphylococcal cell-wall peptidoglycans and proves very effective in combatting methicillin-resistant Staphylococcus aureus. Here, we revealed the functional importance of two loop residues, Tyr in loop 1 and Asn in loop 4, which are highly conserved among the M23 endopeptidase family and are found close to the Zn-coordinating active site. Detailed analyses of the binding groove architecture together with protein-ligand docking showed that these two loop residues potentially interact with the docked ligand-pentaglycine. Ala-substituted mutants (Y270A and N372A) were generated and over-expressed in Escherichia coli as a soluble form at levels comparable to the wild type. A drastic decrease in staphylolytic activity against S. aureus was observed for both mutants, suggesting an essential role of the two loop residues in lysostaphin function. Further substitutions with an uncharged polar Gln side-chain revealed that only the Y270Q mutation caused a dramatic reduction in bioactivity. In silico predicting the effect of binding site mutations revealed that all mutations displayed a large ΔΔG value, signifying requirements of the two loop residues for efficient binding to pentaglycine. Additionally, MD simulations revealed that Y270A and Y270Q mutations induced large flexibility of the loop 1 region, showing markedly increased RMSF values. Further structural analysis suggested that Tyr conceivably participated in the oxyanion stabilization of the enzyme catalysis. Altogether, our present study disclosed that two highly conserved loop residues, loop 1-Tyr and loop 4-Asn, located near the lysostaphin active site are crucially involved in staphylolytic activity toward binding and catalysis of pentaglycine cross-links.