Internally quenched peptides for the study of lysostaphin: an antimicrobial protease that kills Staphylococcus aureus

Abstract

Lysostaphin (EC. 3.4.24.75) is a protein secreted by Staphylococcus simulans biovar staphylolyticus and has been shown to be active against methicillin resistant S. aureus (MRSA). The design and synthesis of three internally quenched substrates for lysostaphin based on the peptidoglycan crossbridges of S. aureus, and their use in fluorescence resonance energy transfer (FRET) assays is reported. These substrates enabled the gathering of information about the endopeptidase activity of lysostaphin and the effect that mutations have on its enzymatic ability. Significant problems with the inner filter effect and substrate aggregation were encountered; their minimisation and the subsequent estimation of the kinetic parameters for the interaction of lysostaphin with the substrates is described, as well as a comparison of substrates incorporating two FRET pairs: Abz–EDDnp and DABCYL–EDANS. In addition to this, the points of cleavage caused by lysostaphin in Abz-pentaglycine-EDDnp have been determined by HPLC and mass spectrometry analysis to be between glycines 2 and 3 (∼60%) and glycines 3 and 4 (∼40%).