Michaelis-Menten Kinetics for Determining Enzymatic Activity of Lysostaphin

Surovtsev, V. I.; Fedorov, T. V.; Borozdina, M. A.

doi:10.1023/b:biry.0000040199.64244.b4

Cited by 3 publications

(5 citation statements)

References 7 publications

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“…Fig. 5 shows the emission spectra obtained from 10 lM solutions of 14 and 17, and 1 lM solutions of N-(2-aminobenzoyl)triglycine (3) and (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid) diglycine (18). It is clear that DABCYL quenches the emission from EDANS more efficiently than EDDnp does the emission of Abz.…”

Section: Scheme 3 Illustrates the Solid Phase-based Synthetic Route T...mentioning

confidence: 99%

“…6 Spectrophotometric variations on this method have also been reported 17 and used to examine the Michaelis-Menten kinetics of the interaction of lysostaphin with whole cells, leading to the determination of the specific activity of the enzyme. 18 Other spectrophotometric assays described include a dye-release assay developed by Zhou et al, 19 which gave results that were more reproducible than the turbidometric method. Kline and co-workers developed a TNBS-based colorimetric micro titre plate assay that monitored the hydrolysis of N-acetylated hexaglycine by lysostaphin.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2006

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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%).

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Section: Scheme 3 Illustrates the Solid Phase-based Synthetic Route T...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2006

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“…Using previously described pseudo-Michaelis-Menten methods (Scanlon et al 2010; Surovtsev et al 2004), we leveraged our fluorescence kinetic assay to study S. aureus lysis rates in greater detail. As part of optimizing the assay conditions, measurements were initially made at two different enzyme concentrations: 200 ng/ml and 400 ng/ml (equating to 7.4 and 14.8 nM for LST and SsaALP-LBP, and 6.5 nM and 12.9 nM for LytO-LBP).…”

Section: Resultsmentioning

confidence: 99%

Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity

Osipovitch

Therrien

Griswold

2015

Appl Microbiol Biotechnol

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Staphylococcus aureus is a dangerous bacterial pathogen whose clinical impact has been amplified by the emergence and rapid spread of antibiotic resistance. In the search for more effective therapeutic strategies, great effort has been placed on the study and development of staphylolytic enzymes, which benefit from high potency, activity towards drug-resistant strains, and a low inherent susceptibility to emergence of new resistance phenotypes. To date, the majority of therapeutic candidates have derived from either bacteriophage or environmental competitors of S. aureus. Little to no consideration has been given to cis-acting autolysins that represent key elements in the bacterium's endogenous cell wall maintenance and recycling machinery. In this study, five putative autolysins were cloned from the S. aureus genome, and their activities were evaluated. Four of these novel enzymes, or component domains thereof, demonstrated lytic activity towards live S. aureus cells, but their potencies were 10s to 1000s of times lower than that of the well-characterized therapeutic candidate lysostaphin. We hypothesized that their poor activities were due in part to suboptimal cell wall targeting associated with their native cell wall binding domains, and we sought to enhance their antibacterial potential via chimeragenesis with the peptidoglycan binding domain of lysostaphin. The most potent chimera exhibited a 140-fold increase in lytic rate, bringing it within 8-fold of lysostaphin. While this enzyme was sensitive to certain biologically relevant environmental factors and failed to exhibit a measurable minimal inhibitory concentration, it was able to kill lysostaphin-resistant S. aureus and ultimately proved active in lung surfactant. We conclude that the S. aureus proteome represents a rich and untapped reservoir of novel antibacterial enzymes, and we demonstrate enhanced bacteriolytic activity via improved cell wall targeting of autolysin catalytic domains.

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“…Atomic structures are available for both the inactive12 and active13 forms of the Zn 2+ metallopeptidase LytM of Staphylococcus aureus , a homolog of lysostaphin of Staphylococcus simulans . Lysostaphin is a well-studied metallopeptidase that cleaves the pentaglycine cross-link of the staphylococcal cell wall during autolysis14–19. The atomic structures of the lysostaphin homolog Ale-1 of Staphylococcus capitis 20 and LAS protein MepA of Escherichia coli 21 have also been determined.…”

Section: Introductionmentioning

confidence: 99%

“…The atomic structures of the lysostaphin homolog Ale-1 of Staphylococcus capitis 20 and LAS protein MepA of Escherichia coli 21 have also been determined. However, the precise molecular mechanism for catalysis by the conserved amino acids within LytM, lysostaphin and the lysostaphin-like enzymes remains unknown14, 18. Models and cocrystallized structures of lysostaphin-like peptidases with bound substrates are also not available.…”

Section: Introductionmentioning

confidence: 99%

Shared Catalysis in Virus Entry and Bacterial Cell Wall Depolymerization

Cohen

Sham

Haugstad

et al. 2009

Journal of Molecular Biology

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Summary Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active site residues and a structural pattern of β-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage φ29. gp13 activity on PG and muropeptides was assayed using high performance liquid chromatography, and gp13 was found to be a D,D-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile bond activation and His247 is oriented to mediate nucleophile generation. This is the first model of a Zn2+-metallopeptidase and its substrate to our knowledge. Residue Asp195 of gp13 was found to be critical for Zn2+-binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle induced X-ray emission spectroscopy showed that the general protein folding and Zn2+-binding was maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model where the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage entry enzymes have a shared chemical mechanism of action.

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Michaelis-Menten Kinetics for Determining Enzymatic Activity of Lysostaphin

Cited by 3 publications

References 7 publications

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

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

Discovery of novel S. aureus autolysins and molecular engineering to enhance bacteriolytic activity

Shared Catalysis in Virus Entry and Bacterial Cell Wall Depolymerization

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