Kinetic Investigation of the Staphylococcal Protease-Catalyzed Hydrolysis of Synthetic Substrates (original) (raw)
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2 Proteases of Staphylococcus aureus – from structure and function to practical application
2008
Proteases are of significant importance for the virulence of S. aureus-a human pathogen causing a wide range of diseases [1, 2]. Proteases of three different catalytic classes are found among the secreted proteins. They are encoded by genes organized in four operons: ssp (encoding the SspA serine and SspB cysteine protease and SspC cysteine protease inhibitor); scp (endoding the ScpA cysteine protease and ScpB cysteine protease inhibitor); spl (the claster of six serine protease-like genes which encode for putative proteases) and aur (encoding metalloprotease and protein of unknown function) [1]. We isolated and characterized several native S. aureus secreted proteases. Using heterologous expression system we obtained recombinant proteins SspC and ScpB that function as endogenous and very specific inhibitors of cysteine protease SspB and ScpA (staphopains), respectively [3, 4]. To date, four members of homologous inhibitors constituting a novel family (staphostatins) have been described [5]. Representatives were structurally characterized in detail by NMR and X-ray crystallography [6, 7]. Although staphopains belong to clan CA of papain-like proteases, staphostatins inhibit exclusively the staphylococcal enzymes and not other clan members tested. Unlike the previously known cysteine protease inhibitors, the staphostatin polypeptide chain spans the active site of the protease in a manner similar to the substrate. The inhibitor escapes cleavage due to the distinct deposition of the P1 glycine residue thus resembling "standard mechanism" serine protease inhibitors. From the six serine protease-like (spl) genes encomposed in one operon-three (SplA, B and C) have been already efficiently expressed and characterized [8, 9]. The enzymes show restricted substrate specificity similar to that of the V8 protease and epidermolytic toxins. The mechanism of unusual activation of SplB protease as well as practical application in recombinant protein technology will be discussed.
Substrate specificity of Staphylococcus aureus cysteine proteases – Staphopains A, B and C
Biochimie, 2012
Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2eGlyYAla(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.
Journal of Applied Microbiology, 2013
Aims: The purpose of this study was to evaluate the antimicrobial efficacy of five different proteases belonging to two different families on Staphylococcus aureus and Staphylococcus epidermidis strains. Methods and Results: We used three serine proteases and two metalloproteases in single species biofilm formation assays and in human cell invasion processes. Following each protease incubation with bacterial cells, surface protein patterns were analysed by SDS-PAGE and zymography. Some differently expressed proteins were identified by mass spectrometry. Conclusions: The effect of tested proteases on biofilm formation was not related to the protease category but was strain-dependent and was related to the biofilm formation capacity of each staphylococcal strain. Some proteases showed a nonspecific and indiscriminate effect on surface proteins, while others induced a discrete and reproducible action on protein profiles. Significance and Impact of the Study: The inhibition of the surface-related virulence factors is a promising avenue to overcome persistent infections caused by bacterial biofilms. To this end, we show here that proteases, in particular the metalloprotease serratiopeptidase, can interfere with adhesion and invasion of eukaryotic cells and biofilm formation in staphylococci and their use could represent a viable treatment for the development of novel combination therapies.
Indian journal of biochemistry & biophysics
Proenzymes with various lengths of propeptides have been observed in GluV8 from Staphylococcus aureus and GluSE from S. epidermidis. However, the production mechanism of these proenzymes and roles of truncated propeptides have yet to be elucidated. Here we demonstrate that shortening of propeptide commonly occurs in an auto-catalytic manner in GluV8-family members, including those from coagulase negative Staphylococci and Enterococcus faecalis. Accompanied with propeptide shortening, the pro-mature junction (Asn/Ser_1-Val1) becomes more susceptible towards the hetero-catalytic maturation enzymes. The auto-catalytic propeptide truncation is not observed in Ser169Ala inert molecules of GluV8-family members. A faint proteolytic activity of proenzymes from Staphylococcus caprae and E. faecalis is detected. In addition, proteolytic activity of proenzyme of GluV8 carrying Arg-3AlaAsn.1 is demonstrated with synthetic peptide substrates LLE/Q-MCA. These results suggest that GluV8-family pro...
Biological Chemistry, 2000
Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE and GluSW, respectively. The order of their protease activities was GluSE<GluSW<<GluV8. The present study investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference was primarily attributed to amino acids at residues 170-195, which defined the intrinsic protease activity, and additionally to residues 119-169, which affected the proteolysis sensitivity. Among nine substitutions present in residues 170-195 of the three proteases, the substitutions at positions 185, 188 and 189 were responsible for the changes in their activities; and the combination of W185, V188 and P189, which naturally occurred on GluV8, exerted the highest protease activity. Among them, W185 and P189 were indispensable for the full activity; but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appeared to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore, defined the K m values of the proteases. This study also describes the way to produce a chimeric form of GluSE and GluV8 that was resistant to proteolysis, and therefore, possessed activity 4-fold higher than that of the wild-type recombinant GluV8.
Pakistan journal of zoology
In this study, the protease producing bacterium was isolated from chicken waste and characterized as Staphylococcus aureus through 16S rRNA ribotyping. The protease from S. aureus S-2 showed maximum activity of 360 U/mL. S. aureus S-2 showed optimum growth at 37ºC and pH 7. S. aureus S-2 was able to grow in 1% gram pulse, mung beans and hydrolyzed casein but the maximum growth of the organism was supported by 1% hydrolyzed casein. A comparison of intracellular and extracellular enzyme activity showed that the predominant form of the enzyme was extracellular. The optimal pH and temperature for the protease activity were 8.0 and 50ºC, respectively. The enzyme was active at a broad range of pH (5-9) and temperatures (30-90ºC). The molecular mass of the enzyme was found to be 30 kDa in SDS-polyacrylamide gel electrophoresis. S. aureus S-2 can be exploited for biotechnological and industrial applications.
Biochemistry, 1993
The hydrolysis of single-stranded DNA catalyzed by wild-type staphylococcal nuclease (SNase) and two mutants has been studied as a function of both pH and solvent viscosity. The kat for wild-type SNase increases with pH; the slope of the plot of log kat us pH = 0.45 f 0.01. The dependence of k,t/Km on pH for wild-type SNase is biphasic with a break at pH -8: for pH 1 8 , the plot of log kat us pH is linear with a slope = 1.20 f 0.06; for pH 1 8 , the slope = 0.00 f 0.04. The dependencies of both kat and k,t/Km on solvent viscosity are also pH-dependent: below pH 7.3, both kinetic parameters are independent of solvent viscosity; above pH 7.3, both are inversely proportional to solvent viscosity. Thus, at pH 9.5, where SNase is routinely assayed, the rate-determining steps for both kat and kat/& are external steps (product dissociation for kat and substrate binding for kat/Km) and not an internal step (e.g., hydrolysis of the phosphodiester bond). We have also studied the E43D mutant in which the putative active-site general basic catalyst Glu-43 is replaced with Asp. From pH 7.5 to pH 9.5, both log kat and log (kat/K,) are directly proportional to pH (slopes = 1.01 f 0.03 and 0.95 f 0.04, respectively) and independent of solvent viscosity. At pH 9.5, the rate-determining step is an internal step. Since the rate-determining steps differ at pH values 27.5, we conclude that comparisons of the numerical values of the kinetic parameters of active-site mutants of SNase with those of wild-type SNase are mechanistically uninformative at pH values where the kats are maximal. We have begun to localize the structural elements responsible for the slow external step (product dissociation) in the kcat for wild-type SNase by characterizing a mutant (PVN ASNase) in which residues 4 4 4 9 of the 8-loop (residues 43-52) have been deleted to generate a new solvent-exposed /%turn; in addition, Gly-50 and Val-5 1 have been replaced with Val and Asn, respectively.
Journal of Molecular Biology, 2008
Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the threedimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.
Biochemistry, 1975
Proton magnetic resonance spectra of staphylococcal protease, a serine protease from Staphylococcus aureus, strain V8, are presented. Initial proton spectra were obtained at 220 MHz, and more detailed studies of the aromatic region were carried out by correlation spectroscopy a t 250 MHz. The overall spectrum bears a close resemblance to one calculated from the sum of spectra of the component amino acids. Chemical shifts of the three tyrosine, four phenylalanine, and three histidine residues appear to be equivalent a t pH 3.7 and 8.5 indicating that they are all in normal chemical environments in the enzyme. The staphylococcal protease contains a large number of slowly exchanging protons. In fact, interpretable spectra of the aromatic region were obtained only after extensive exchange of N-H groups with deuterium from the DzO solvent. Proton magnetic resonance titration studies of the three histidine residues indicate that these have normal chemical shifts and pK' values.
Purification and characterization of a serine alkaline protease from Bacillus clausii GMBAE 42
Journal of Industrial Microbiology & Biotechnology, 2005
An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37°C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH4)2SO4 precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60°C; however, it is shifted to 70°C after addition of 5 mM Ca2+ ions. The enzyme was stable between 30 and 40°C for 2 h at pH 10.5; only 14% activity loss was observed at 50°C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0–12.2 range for 24 h at 30°C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol−1 (44.30 kJ mol−1). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30°C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3′-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k cat value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K m and k cat values were estimated at 0.655 μM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21×103 min−1, respectively.