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

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(approximately 60%) and glycines 3 and 4 (approximately 40%).

[1]  D. Ullmann,et al.  Design and synthesis of fluorogenic trypsin peptide substrates based on resonance energy transfer. , 1998, Analytical biochemistry.

[2]  L. Heath,et al.  The lysostaphin endopeptidase resistance gene (epr) specifies modification of peptidoglycan cross bridges in Staphylococcus simulans and Staphylococcus aureus , 1995, Applied and environmental microbiology.

[3]  R. Knox,et al.  “Celbenin” - resistant Staphylococci , 1961 .

[4]  R. Zhou,et al.  A dye release assay for determination of lysostaphin activity. , 1988, Analytical biochemistry.

[5]  I. Márová,et al.  Modified simplified method for isolation of lysostaphin from the culture filtrate ofStaphylococcus staphylolyticus , 2008, Folia Microbiologica.

[6]  S. Walsh,et al.  Lysostaphin-Coated Catheters Eradicate Staphylococccus aureus Challenge and Block Surface Colonization , 2004, Antimicrobial Agents and Chemotherapy.

[7]  J. Robinson,et al.  Relationship between lysostaphin endopeptidase production and cell wall composition in Staphylococcus staphylolyticus , 1979, Journal of bacteriology.

[8]  S. A. Kline,et al.  A colorimetric microtiter plate assay for lysostaphin using a hexaglycine substrate. , 1994, Analytical biochemistry.

[9]  E. Katchalski‐Katzir,et al.  Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes. , 1979, Analytical biochemistry.

[10]  S. V. Sinitsyn,et al.  H2O2-Induced Inhibition of Photosynthetic O2 Evolution by Anabaena variabilis Cells , 2004, Biochemistry (Moscow).

[11]  L. S. Harrow.  Organic Syntheses, Collective , 1955 .

[12]  M. Sugai,et al.  Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1 , 1997, Journal of bacteriology.

[13]  L. Juliano,et al.  Internally quenched fluorogenic substrates for angiotensin I-converting enzyme. , 1999, Journal of hypertension.

[14]  L. Juliano,et al.  Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides. , 2001, Analytical biochemistry.

[15]  S. G. Odintsov,et al.  Latent LytM at 1.3A resolution. , 2004, Journal of molecular biology.

[16]  M. Bochtler,et al.  Crystal structures of active LytM. , 2005, Journal of molecular biology.

[17]  N. Thornberry,et al.  Synthesis of a fluorogenic interleukin-1 beta converting enzyme substrate based on resonance energy transfer. , 1994, Peptide research.

[18]  S. Walsh,et al.  Lysostaphin Cream Eradicates Staphylococcus aureus Nasal Colonization in a Cotton Rat Model , 2003, Antimicrobial Agents and Chemotherapy.

[19]  Mary Jane Ferraro,et al.  Linezolid resistance in a clinical isolate of Staphylococcus aureus , 2001, The Lancet.

[20]  B. Dunn,et al.  Peptide Synthesis Protocols , 1994 .

[21]  Gary T. Wang,et al.  Novel fluorogenic substrates for assaying retroviral proteases by resonance energy transfer. , 1990, Science.

[22]  R. Jayaswal,et al.  Molecular cloning, sequencing, and expression of lytM, a unique autolytic gene of Staphylococcus aureus , 1997, Journal of bacteriology.

[23]  D. Ohman,et al.  A continuous spectrophotometric assay for Pseudomonas aeruginosa LasA protease (staphylolysin) using a two-stage enzymatic reaction. , 2004, Analytical biochemistry.

[24]  Th. Förster.  Zwischenmolekulare Energiewanderung und Fluoreszenz , 1948 .

[25]  O. Schneewind,et al.  Cross-Linked Peptidoglycan Mediates Lysostaphin Binding to the Cell Wall Envelope of Staphylococcus aureus , 2006, Journal of bacteriology.

[26]  V. Surovtsev,et al.  Michaelis-Menten Kinetics for Determining Enzymatic Activity of Lysostaphin , 2004, Biochemistry (Moscow).

[27]  I. Márová,et al.  Spectrophotometric detection of bacteriolytic activity of diluted lysostaphin solutions , 2008, Folia Microbiologica.

[28]  M. Jevons,et al.  Cellbenin-resistant staphylococci. , 1961 .

[29]  C. Tellier,et al.  Synthesis of 15 Enriched Phospholipids by Transphosphatidylation , 1985 .

[30]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[31]  D. Boger,et al.  Key analogs of the tetrapeptide subunit of RA-VII and deoxybouvardin. , 1995, Bioorganic & medicinal chemistry.

[32]  F. Tenover,et al.  Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. , 1997, The Journal of antimicrobial chemotherapy.

[33]  W. Schröder,et al.  Site-Specific Serine Incorporation by Lif and Epr into Positions 3 and 5 of the Staphylococcal Peptidoglycan Interpeptide Bridge , 2000, Journal of bacteriology.

[34]  C. Schindler,et al.  LYSOSTAPHIN: A NEW BACTERIOLYTIC AGENT FOR THE STAPHYLOCOCCUS. , 1964, Proceedings of the National Academy of Sciences of the United States of America.

[35]  F. Götz,et al.  Studies on prolysostaphin processing and characterization of the lysostaphin immunity factor (Lif) of Staphylococcus simulans biovar staphylolyticus , 1997, Molecular microbiology.

[36]  M. Sugai,et al.  Cell Wall-targeting Domain of Glycylglycine Endopeptidase Distinguishes among Peptidoglycan Cross-bridges* , 2006, Journal of Biological Chemistry.