Conserved loop residues−Tyr<sup>270</sup> and Asn<sup>372</sup> near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis
Issued Date
2023-08-06
Resource Type
ISSN
0006291X
eISSN
10902104
Scopus ID
2-s2.0-85160055535
Journal Title
Biochemical and Biophysical Research Communications
Volume
668
Start Page
111
End Page
117
Rights Holder(s)
SCOPUS
Bibliographic Citation
Biochemical and Biophysical Research Communications Vol.668 (2023) , 111-117
Suggested Citation
Charoenjotivadhanakul S., Sakdee S., Imtong C., Li H.C., Angsuthanasombat C. Conserved loop residues−Tyr<sup>270</sup> and Asn<sup>372</sup> near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis. Biochemical and Biophysical Research Communications Vol.668 (2023) , 111-117. 117. doi:10.1016/j.bbrc.2023.05.085 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/82972
Title
Conserved loop residues−Tyr<sup>270</sup> and Asn<sup>372</sup> near the catalytic site of the lysostaphin endopeptidase are essential for staphylolytic activity toward pentaglycine binding and catalysis
Other Contributor(s)
Abstract
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, Tyr270 in loop 1 and Asn372 in loop 4, which are highly conserved among the M23 endopeptidase family and are found close to the Zn2+-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 ΔΔGbind 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 Tyr270 conceivably participated in the oxyanion stabilization of the enzyme catalysis. Altogether, our present study disclosed that two highly conserved loop residues, loop 1-Tyr270 and loop 4-Asn372, located near the lysostaphin active site are crucially involved in staphylolytic activity toward binding and catalysis of pentaglycine cross-links.
