Liberation of surface-located penicillinase from Staphylococcus aureus

1. Growth of Staphylococcus aureus (8325; αi⁻p⁺), constitutive for the production of penicillinase, in CY medium results in about 40% of the enzyme being free in the medium. By modifying the medium, 98% of the enzyme remains cell-bound. 2. Part of this is bound ionically to the surface of the cell wall and may be liberated instantaneously by certain inorganic anions. Maximum liberation was achieved with either phosphate or arsenate, both of which showed marked pH-dependence. 3. Polyanions that do not penetrate the cell wall, such as heparin, RNA and dextran sulphate, are also effective in liberating penicillinase. 4. Polyanions added to the growth medium prevent the appearance of ionically bound penicillinase owing to their strong affinity for the sites on the cell wall required for binding of the enzyme.     

The amino acid sequence of Staphylococcus aureus penicillinase.

The amino acid sequence of the penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus strain PC1 was determined. The protein consists of a single polypeptide chain of 257 residues, and the sequence was determined by characterization of tryptic, chymotryptic, peptic and CNBr peptides, with some additional evidence from thermolysin and S. aureus proteinase peptides. A mistake in the preliminary report of the sequence is corrected; residues 113-116 are now thought to be -Lys-Lys-Val-Lys- rather than -Lys-Val-Lys-Lys-. Detailed evidence for the amino acid sequence has been deposited as Supplementary Publication SUP 50056 (91 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.     

Characteristics of penicillinase plasmids from Staphylococcus aureus strains

Penicillinase plasmids are present in most MRSA strains. They are very varying in their genotype and phenotype they confer. Penicillinase plasmids were transduced from 80 hospital MRSA strains to NCTC 8325 and the phenotype as well as the incompatibility group of plasmid were determined. Resistance to cadmium (high and low level), resistance to organic and nonorganic mercury compounds, arsenate/arsenite/antimonium resistance, resistance to bismuth and hypersensitivity to bismuth, resistance to macrolides as well as beta-lactamase production and its inductibility were checked. Among the examined strains 20 different phenotypes of penicillinase plasmids were found. Patterns of penicillinase plasmids were compared to DNA patterns of the investigated strains after digestion with SmaI and separation in pulsed field electrophoresis (PFGE). It was shown that strains with the same PFGE pattern often differ in the type of their penicillinase plasmid. Determining of penicillinase plasmid phenotype could be useful in differentiating S. aureus strains sharing the same pattern of PFGE.     

Isolation of a serologically different compact-colony-forming active substance from strains of Staphylococcus aureus

To observe the possible serological heterogeneity of compact-colony-forming active substance (CCFAS), heat-killed vaccines were prepared by two strains of Staphylococcus aureus, strains SMU 1-46 and SMU 7931, cultured in 0.03 M trishydrochloride-buffered brain heart infusion, pH 8.4. After immunization with the vaccine in rabbits, antibody responses were observed during a period of six weeks after the immunization either by homologous and heterologous organisms using alkaline serum-soft agar technique. The results showed that remarkable antibody production was shown only against homologous strain, but not against heterologous strain. The antibodies were absorbed out only with highly purified preparation of CCFAS extracted from homologous strain and not with heterologous CCFAS. Differences of the major chemical composition of the substances showed that highly purified CCFAS extracted from strain SMU 7931 contained 2.84 and 2.04 times higher amounts of galactose and 2-amino-2-deoxy-D-galacturonic acid than those of CCFAS obtained from strain SMU 1-46.     

Probing the active site tryptophan of Staphylococcus aureus thioredoxin with an analog

Genetically encoded non-canonical amino acids are powerful tools of protein research and engineering; in particular they allow substitution of individual chemical groups or atoms in a protein of interest. One such amino acid is the tryptophan (Trp) analog 3-benzothienyl-l-alanine (Bta) with an imino-to-sulfur substitution in the five-membered ring. Unlike Trp, Bta is not capable of forming a hydrogen bond, but preserves other properties of a Trp residue. Here we present a pyrrolysyl-tRNA synthetase-derived, engineered enzyme BtaRS that enables efficient and site-specific Bta incorporation into proteins of interest in vivo. Furthermore, we report a 2.1 Å-resolution crystal structure of a BtaRS•Bta complex to show how BtaRS discriminates Bta from canonical amino acids, including Trp. To show utility in protein mutagenesis, we used BtaRS to introduce Bta to replace the Trp28 residue in the active site of Staphylococcus aureus thioredoxin. This experiment showed that not the hydrogen bond between residues Trp28 and Asp58, but the bulky aromatic side chain of Trp28 is important for active site maintenance. Collectively, our study provides a new and robust tool for checking the function of Trp in proteins.     

Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid

Previously described penicillinase-constitutive mutations in Staphylococcus aureus are caused by genetic lesions in a regulator gene (or genes) on the penicillinase plasmid in close linkage to the structural gene. This report describes a new class (R2(-)) of penicillinase-constitutive mutants of S. aureus unlinked to the plasmid. By transductional analysis, the penicillinase plasmids in these mutants were wild type. Wild-type plasmids transduced into penicillinase-negative (plasmid loss) derivatives of R2(-) mutants produced penicillinase constitutively in amounts comparable to a fully induced culture of the wild-type strain. Penicillinase production in R2(-) mutants was maximal at 30 to 32 C and was much reduced at 40 C.     

The reversible deactivation of beta-lactamase from Staphylococcus aureus by quinacillin and cephaloridine and its modification by antibodies

The effect of antibody on the reversible deactivation of the beta-lactamase (penicillin amino-beta-lactamhydrolase, EC 3.5.2.6) from Staphylococcus aureus has been studied using quinacillin and cephaloridine as substrates. The latter has been shown to exhibit the characteristics of an A-type substrate Citri, N., Samuni, A. and Zyk, N. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 1048-1052) and reversibly to lower the activity of the enzyme towards benzylpenicillin in a manner analogous to quinacillin. Both divalent and monovalent antibodies reduce the activity of the lactamase to 60% of the native value in the absence of substrate. The reduction by monovalent antibody is slow (t1/2 approximately equal to 25 min). Both divalent and monovalent antibodies modify the time-course of reversible deactivation independently of being added before or subsequent to deactivation by substrate. The full recovery of activity is delayed in the case of quinacillin and accelerated for cephaloridine. The activity against benzylpenicillin in the deactivated states is unaffected. These effects are shown to reflect the changed rates of hydrolysis of the two substrates in the presence of antibody. The effect of antibody is mediated by minor conformational change. Continuous assays for following the hydrolysis of quinacillin and cephaloridine by optical rotation are reported.     

Preferential nitration with tetranitromethane of a specific tyrosine residue in penicillinase from Staphylococcus aureus PCl. Evidence that the preferentially nitrated residue is not part of the active site but that loss of activity is due to intermolecular cross-linking.

1. Nitration of tyrosine residues of staphylococal penicillinase was accompanied by a partial loss of enzymic activity, which was not readily explained by nitration of a single residue. 2. Loss of activity correlated with low recovery of tyrosine plus nitrotyrosine, which was consistent with cross-linking. 3. The fraction of treated enzyme that was eluted from Sephadex G-75 earlier than native penicillinase was similar to the fraction of enzyme activity lost. Protein eluted in positions corresponding to monomer, dimer and higher oligomers respectively showed major bands in corresponding positions in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicating that the increase in molecular weight was due to intermolecular cross-linking. Monomeric enzyme containing up to 4 mol of nitrotyrosine/mol retained full catalytic activity. Dimeric enzyme retained 50% of normal activity, whereas higher oligomers retained an average of 8-15% of normal activity. 4. Monomeric enzyme isolated after treatment with equimolar tetranitromethane was nitrated predominantly at tyrosine-72.5. Reaction of reduced nitrated monomer with 1,5-difluoro-2,4-dinitrobenzene gave a monomeric, apparently cross-linked product with full catalytic activity. 6. It is concluded that tyrosine-72 plays no part in the active site. Its preferential nitration may be due to its being insufficiently exposed to be available for intermolecular cross-linking. This poperty may make it useful for attachment of a reporter group.     

Crystal structure of Sa240: a ribose pyranase homolog with partial active site from Staphylococcus aureus

Ribose is transported into cells in its pyranose form and must be rearranged to its furanose form for further utilization. Ribose pyranase RbsD catalyzes the conversion of ribose from the pyranose to furanose form. This is the key step for substrate supply to ribokinase RbsK, which converts ribose to ribose-5-phosphate for further metabolism. Sequence analysis indicated Sa240 from Staphylococcus aureus was a ribose pyranase homolog. Here we showed that Sa240 formed dimeric structure both in solution and in crystal. S240-ribose complex structure showed a ribose binding site formed by an incomplete active site compared with RbsD. Because the catalytic activity of ribose pyranase depends on its oligomeric state, we propose Sa240 is catalytically inactive in its dimeric structure.     

The conserved active site proline determines the reducing power of Staphylococcus aureus thioredoxin

Nature uses thioredoxin-like folds in several disulfide bond oxidoreductases. Each of them has a typical active site Cys-X-X-Cys sequence motif, the hallmark of thioredoxin being Trp-Cys-Gly-Pro-Cys. The intriguing role of the highly conserved proline in the ubiquitous reducing agent thioredoxin was studied by site-specific mutagenesis of Staphylococcus aureus thioredoxin (Sa_Trx). We present X-ray structures, redox potential, pK(a), steady-state kinetic parameters, and thermodynamic stabilities. By replacing the central proline to a threonine/serine, no extra hydrogen bonds with the sulphur of the nucleophilic cysteine are introduced. The only structural difference is that the immediate chemical surrounding of the nucleophilic cysteine becomes more hydrophilic. The pK(a) value of the nucleophilic cysteine decreases with approximately one pH unit and its redox potential increases with 30 mV. Thioredoxin becomes more oxidizing and the efficiency to catalyse substrate reduction (k(cat)/K(M)) decreases sevenfold relative to wild-type Sa_Trx. The oxidized form of wild-type Sa_Trx is far more stable than the reduced form over the whole temperature range. The driving force to reduce substrate proteins is the relative stability of the oxidized versus the reduced form Delta(T(1/2))(ox/red). This driving force is decreased in the Sa_Trx P31T mutant. Delta(T(1/2))(ox/red) drops from 15.5 degrees C (wild-type) to 5.8 degrees C (P31T mutant). In conclusion, the active site proline in thioredoxin determines the driving potential for substrate reduction.     

Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.     

Kinetics and active site dynamics of Staphylococcus aureus arsenate reductase

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular arsenate(V) to the more toxic arsenite(III), which is subsequently extruded from the cell. It couples to thioredoxin, thioredoxin reductase and NADPH to be enzymatically active. ArsC is extremely sensitive to oxidative inactivation, has a very dynamic character hampering resonance assignments in NMR and produces peculiar biphasic Michaelis-Menten curves with two V(max) plateaus. In this study, methods to control ArsC oxidation during purification have been optimized. Next, application of Selwyn's test of enzyme inactivation was applied to progress curves and reveals that the addition of tetrahedral oxyanions (50 mM sulfate, phosphate or perchlorate) allows the control of ArsC stability and essentially eliminates the biphasic character of the Michaelis-Menten curves. Finally, 1H-15N HSQC NMR spectroscopy was used to establish that these oxyanions, including the arsenate substrate, exert their stabilizing effect on ArsC through binding with residues located within a C-X5-R sequence motif, characteristic for phosphotyrosine phosphatases. In view of this need for a tetrahedral oxyanion to structure its substrate binding site in its active conformation, a reappraisal of basic kinetic parameters of ArsC was necessary. Under these new conditions and in contrast to previous observations, ArsC has a high substrate specificity, as only arsenate could be reduced ( Km=68 microM, k(cat)/ Km =5.2 x 10(4 )M-1s-1), while its product, arsenite, was identified as a mixed inhibitor ( K*iu=534 microM, K*ic=377 microM).     

Chemical and electron microscopic studies of factors associated with the release of penicillinase from Staphylococcus aureus

The effect of various factors such as sodium chloride, sodium citrate, pH, buffers, and enzymatic and physical disruption of cells on the release of penicillinase by Staphylococcus aureus ATCC 14458 was investigated. Penicillinase was measured at selected time intervals from supernates of cultures grown in Antibiotic Medium 3 broth containing various concentrations of salts or buffers or from supernates of cultures treated with lysostaphin and subsequently disrupted by French press treatment.Incubation of cells with media containing either sodium chloride (5, 10, and 15%), sodium citrate (5 and 10%), or organic buffers (Tris-HC1, 2.5, 5.0, and 7.5%; BES, 10 and 20%) resulted in a significant stimulation of the release of penicillinase when compared to control cells. It was also observed that pH 7.0–7.5 was optimal for penicillinase activity and release. From studies of enzymatic and mechanical disruption of cells, it was observed that an increase in ionic strength of the suspending medium to certain optimal levels appeared to stimulate the conversion of penicillinase to an extracellular form.Electron microscopic studies revealed that a large number of mesosomal vesicles seemed to be present in cells incubated for 4 hours in media containing various concentrations of sodium chloride. It is proposed that either appearance of vesicles or convolution of cell membrane, which may be caused by further synthesis of new membrane, is involved in stimulation of the synthesis and release of membrane-bound penicillinase.     

Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.