Accumulation of acyl-enzyme intermediates during turnover of penicillins by the class A beta-lactamase of Staphylococcus aureus PC1.
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ABSTRACT:
SUBMITTER: Pratt RF
PROVIDER: S-EPMC1135173 | biostudies-other | 1988 Sep
REPOSITORIES: biostudies-other
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Project description:The pH- and temperature-dependence of steady-state kinetic parameters for 6-beta-(2-furyl)-acryloylamido-penicillanic acid showed it to be a good substrate of staphylococcal PC1 beta-lactamase, and the viscosity-dependence of K(m)/k(cat) indicated that steps up to the formation of the acyl-enzyme were partially diffusion-limited. In the pH range 4-9, a pre-steady-state transient blue shift in the UV absorption spectrum of the bound furyl-acryloylamido chromophore was of constant amplitude and decayed to the spectrum of the product with a first-order rate constant equal to k(cat). The spectrum of the isolated denatured acyl-enzyme was similar to that of the methyl ester of furyl-acryloylpenicilloic acid, pointing to non-covalent interactions with the folded protein, possibly associated with the charge on Glu-166, as the source of the blue-shifted spectrum. Taken together, these results point to a rapid acylation and slower deacylation at Ser-70 and imply that ionization of groups affecting enzyme activity at alkaline pH, for which likely candidates are Lys-73 and Lys-234, affect the rate of deacylation.
| S-EPMC1217229 | biostudies-other
Project description:Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for 'susceptible' MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections.
| S-EPMC7611363 | biostudies-literature
Project description:?-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A ?-lactamases. Widespread resistance to ?-lactam antibiotics currently limits the treatment strategies for Staphylococcus infections. The goals of this study were to determine the binding affinity of BLIP for Staphylococcus aureus PC1 ?-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (K(i)) for PC1 ?-lactamase was measured at 350 nM, and isothermal titration calorimetry experiments indicated a binding constant (K(d)) of 380 nM. Twenty-three residue positions in BLIP that contact ?-lactamase were randomized, and phage display was used to sort the libraries for tight binders to immobilized PC1 ?-lactamase. The BLIP(K74G) mutant was the dominant clone selected, and it was found to inhibit the PC1 ?-lactamase with a K(i) of 42 nM, while calorimetry indicated a K(d) of 26 nM. Molecular modeling studies suggested that BLIP binds weakly to the PC1 ?-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme, where it forms a salt bridge with the BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the PC1(A104E) enzyme binds BLIP with 15-fold greater affinity than wild-type PC1 ?-lactamase. Kinetic measurements indicated similar association rates for all complexes with variation in affinity due to altered dissociation rate constants, suggesting that changes in short-range interactions are responsible for the altered binding properties of the mutants.
| S-EPMC3081586 | biostudies-literature
Project description:The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.
| S-EPMC1138342 | biostudies-other
Project description:The discovery that the mechanism of beta-lactam hydrolysis catalyzed by the class A (active site serine-dependent) beta-lactamases proceeds via an acyl-enzyme intermediate was made thirty years ago. Since this discovery, the active site circumstance that enables acylation of the active site serine and further enables hydrolytic deacylation of the acyl-serine intermediate, has received extraordinary scrutiny. The justification for this scrutiny is the direct relevance of the beta-lactamases to the manifestation of bacterial resistance to the beta-lactam antibiotics, and the subsequent (to the discovery of the beta-lactamase acyl-enzyme) recognition of the direct evolutionary relationship between the serine beta-lactamase acyl-enzyme, and the penicillin binding protein acyl-enzyme that is key to beta-lactam antibiotic activity. This short review describes the early events leading to the recognition that serine beta-lactamase catalysis proceeds via an acyl-enzyme intermediate, and summarizes several of the key mechanistic studies--including infrared spectroscopy, cryoenzymology, beta-lactam design, and x-ray crystallography--that have been exploited to understand this pivotal catalytic intermediate.
| S-EPMC6902449 | biostudies-literature
Project description:Cas9 nuclease is the key effector of type II CRISPR adaptive immune systems found in bacteria. The nuclease can be programmed by a single guide RNA (sgRNA) to cleave DNA in a sequence-specific manner. This property has led to its widespread adoption as a genome editing tool in research laboratories and holds great promise for biotechnological and therapeutic applications. The general mechanistic features of catalysis by Cas9 homologs are comparable; however, a high degree of diversity exists among the protein sequences, which may result in subtle mechanistic differences. S. aureus (SauCas9) and especially S. pyogenes (SpyCas9) are among the best-characterized Cas9 proteins and share ∼17% sequence identity. A notable feature of SpyCas9 is an extremely slow rate of reaction turnover, which is thought to limit the amount of substrate DNA cleavage. Using in vitro biochemistry and enzyme kinetics, we directly compare SpyCas9 and SauCas9 activities. Here, we report that in contrast to SpyCas9, SauCas9 is a multiple-turnover enzyme, which to our knowledge is the first report of such activity in a Cas9 homolog. We also show that DNA cleaved with SauCas9 does not undergo any detectable single-stranded degradation after the initial double-stranded break observed previously with SpyCas9, thus providing new insights and considerations for future design of CRISPR/Cas9-based applications.
| S-EPMC6298560 | biostudies-literature
Project description:It has been shown previously [Faraci & Pratt (1985) Biochemistry 24, 903-910; (1986) Biochemistry 25, 2934-2941; (1986) Biochem. J. 238, 309-312] that certain beta-lactam-processing enzymes form inert acyl-enzymes with cephems that possess good leaving groups at the C-3' position. These inert species arise by elimination of the leaving group from the initially formed and more rapidly hydrolysing acyl-enzyme, which has the 'normal' cephalosporoate structure. The present paper shows that a strong nucleophile, thiophenoxide, can catalyse the re-activation of three examples of these inert acyl-enzymes, generated on reaction of cephalothin and cefoxitin with the PC1 beta-lactamase of Staphylococcus aureus and of cephalothin with D-alanyl-D-alanine transpeptidase/carboxypeptidase of Streptomyces R61. In view of the reversibility of the elimination reaction, demonstrated in model systems [Pratt & Faraci (1986) J. Am. Chem. Soc. 108, 5328-5333], this catalysis is proposed to arise through nucleophilic addition to the exo-methylene carbon atom of the inert acyl-enzyme to regenerate a more rapidly hydrolysing normal cephalosporoate. Strong support for this scenario was obtained through comparison of the kinetics of the catalysed re-activation reaction with those of turnover of the relevant 3'-thiophenoxycephems, thiophenoxycephalothin and thiophenoxycefoxitin. The enzymes appear to stabilize the products of the elimination reaction with respect to the normal cephalosporoate, but more strongly to destabilize the transition states. The effects of other nucleophiles, including cysteine, glycine amide and imidazole, on the above enzymes and on other beta-lactamases can be understood in terms of the model reaction kinetics and thermodynamics.
| S-EPMC1148329 | biostudies-other
Project description:Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small GTPases CDC42, Rac, and RhoB. Actin stress fiber disassembly required for host invasion was attenuated by simvastatin and by the inhibition of phosphoinositide 3-kinase (PI3K) activity. PI3K relies on coupling to prenylated proteins, such as this subset of small GTPases, for access to membrane-bound phosphoinositide to mediate stress fiber disassembly. Therefore, we examined whether simvastatin restricts PI3K cellular localization. In response to simvastatin, the PI3K isoform p85, coupled to these small-GTPases, was sequestered within the cytosol. From these findings, we propose a mechanism whereby simvastatin restricts p85 localization, inhibiting the actin dynamics required for bacterial endocytosis. This approach may provide the basis for protection at the level of the host in invasive infections by S. aureus.
| S-EPMC4482125 | biostudies-literature
Project description:BackgroundMethicillin-sensitive Staphylococcus aureus (MSSA) bacteremia is associated with significant morbidity, mortality, and hospitalization costs. Cefazolin and antistaphylococcal penicillins (ASPs), such as nafcillin, are the preferred treatments for MSSA bacteremia. The aim of this study was to compare the cost-effectiveness of each approach.MethodsWe constructed a decision-analytic model comparing the use of cefazolin with ASPs for the treatment of MSSA bacteremia. Cost-effectiveness was determined by calculating deaths averted and incremental cost-effectiveness ratios (ICERs). Uncertainty was addressed by plotting cost-effectiveness planes and acceptability curves for various willingness-to-pay thresholds.ResultsIn the base-case analysis, the cost associated with the cefazolin strategy was $38 863.1, and the associated probability of survival was 0.91. For the ASP strategy, the cost was $48 578.8, and the probability of survival was 0.81. The incremental difference in cost between the 2 strategies was $9715.7, with hospital length of stay being the main driver of cost, and the incremental difference in effectiveness was 0.10. Overall, cefazolin results in savings of $97 156.8 per death averted (ICER, $-97 156.8/death averted). In the probabilistic analysis, at a willingness-to-pay of $50 000, cefazolin had a 68% chance of being cost-effective compared with ASPs. In cost-effectiveness acceptability curves, the cefazolin strategy was cost-effective in 73.5%-81.8% of simulations compared with ASP for a willingness-to-pay ranging up to $50 000.ConclusionsThe use of cefazolin is a cost-effective strategy for the treatment of MSSA bacteremia and, when clinically appropriate, this strategy results in considerable health care cost-savings.
| S-EPMC8566905 | biostudies-literature
Project description:Neutrophils are indispensable for clearing infections with the prominent human pathogen Staphylococcus aureus. Here, we report that S. aureus secretes a family of proteins that potently inhibits the activity of neutrophil serine proteases (NSPs): neutrophil elastase (NE), proteinase 3, and cathepsin G. The NSPs, but not related serine proteases, are specifically blocked by the extracellular adherence protein (Eap) and the functionally orphan Eap homologs EapH1 and EapH2, with inhibitory-constant values in the low-nanomolar range. Eap proteins are together essential for NSP inhibition by S. aureus in vitro and promote staphylococcal infection in vivo. The crystal structure of the EapH1/NE complex showed that Eap molecules constitute a unique class of noncovalent protease inhibitors that occlude the catalytic cleft of NSPs. These findings increase our insights into the complex pathogenesis of S. aureus infections and create opportunities to design novel treatment strategies for inflammatory conditions related to excessive NSP activity.
| S-EPMC4246989 | biostudies-literature