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Efficient catalysis by beta-lactamase from Staphylococcus aureus PC1 accompanied by accumulation of an acyl-enzyme.

ABSTRACT: 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.

SUBMITTER: Qi X

PROVIDER: S-EPMC1217229 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Accumulation of acyl-enzyme intermediates during turnover of penicillins by the class A beta-lactamase of Staphylococcus aureus PC1.

Project description:The interaction of dansylpenicillin with the class A Staphylococcus aureus PCI beta-lactamase yielded an accumulating intermediate with fluorescence enhanced beyond that of the substrate. Acid quenching of the reaction mixture yielded a denatured enzyme with 1 molar equivalent of dansyl group covalently bound to it. A similar quenching experiment with the PC1 beta-lactamase and [14C]benzylpenicillin yielded an enzyme with 1 molar equivalent of 14C covalently bound. These data indicate that in turnover of S-type penicillins by the PC1 beta-lactamase deacylation is rate-determining. This has not indicate previously been demonstrated for a class A beta-lactamase.

| S-EPMC1135173 | biostudies-other

Trapping the acyl-enzyme intermediate in beta-lactamase I catalysis.

Project description:Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.

| S-EPMC1133516 | biostudies-other

Identification of a ?-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 ?-lactamase.

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.

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Staphylococcus aureus Lipoprotein Induces Skin Inflammation, Accompanied with IFN-?-Producing T Cell Accumulation through Dermal Dendritic Cells.

Project description:Staphylococcus aureus (S. aureus) is a commensal bacteria on the human skin, which causes serious skin inflammation. Several immune cells, especially effector T cells (Teff), have been identified as key players in S. aureus-derived skin inflammation. However, the bacterial component that induces dramatic host immune responses on the skin has not been well characterized. Here, we report that S. aureus lipoprotein (SA-LP) was recognized by the host immune system as a strong antigen, so this response induced severe skin inflammation. SA-LP activated dendritic cells (DCs), and this activation led to Teff accumulation on the inflamed skin in the murine intradermal (ID) injection model. The skin-accumulated Teff pool was established by IFN-?-producing CD4? and CD8?T (Th1 and Tc1). SA-LP activated dermal DC (DDC) in a dominant manner, so that these DCs were presumed to possess the strong responsibility of SA-LP-specific Teff generation in the skin-draining lymph nodes (dLN). SA-LP activated DC transfer into the mice ear, which showed similar inflammation, accompanied with Th1 and Tc1 accumulation on the skin. Thus, we revealed that SA-LP has a strong potential ability to establish skin inflammation through the DC-Teff axis. This finding provides novel insights not only for therapy, but also for the prevention of S. aureus-derived skin inflammation.

| S-EPMC6161079 | biostudies-literature

Clavulanate inactivation of Staphylococcus aureus beta-lactamase.

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.

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The hydrolytic water molecule of Class A β-lactamase relies on the acyl-enzyme intermediate ES* for proper coordination and catalysis.

Project description:Serine-based β-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate β-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the β-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated β-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A β-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi-Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent β-lactamase-mediated antibiotic resistance.

| S-EPMC7311446 | biostudies-literature

Three decades of the class A beta-lactamase acyl-enzyme.

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

Nucleophilic re-activation of the PC1 beta-lactamase of Staphylococcus aureus and of the DD-peptidase of Streptomyces R61 after their inactivation by cephalosporins and cephamycins.

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

Kinetic characterization of the acyl-enzyme mechanism for beta-lactamase I.

Project description:beta-Lactamase I catalyses the hydrolysis of penicillins by an acyl-enzyme mechanism. A procedure was developed for determining the rate constants for the acylation and deacylation steps for the good substrates benzylpenicillin and phenoxymethylpenicillin; this depends on determining the fraction of enzyme that is present as acyl-enzyme in the steady state.

| S-EPMC1135174 | biostudies-other

Acyl-chain selectivity and physiological roles of Staphylococcus aureus fatty acid-binding proteins.

Project description:Fatty acid (FA) kinase produces acyl-phosphate for the synthesis of membrane phospholipids in Gram-positive bacterial pathogens. FA kinase consists of a kinase protein (FakA) that phosphorylates an FA substrate bound to a second module, an FA-binding protein (FakB). Staphylococcus aureus expresses two distinct, but related, FakBs with different FA selectivities. Here, we report the structures of FakB1 bound to four saturated FAs at 1.6-1.93 Å resolution. We observed that the different FA structures are accommodated within a slightly curved hydrophobic cavity whose length is governed by the conformation of an isoleucine side chain at the end of the tunnel. The hydrophobic tunnel in FakB1 prevents the binding of cis-unsaturated FAs, which are instead accommodated by the kinked tunnel within the FakB2 protein. The differences in the FakB interiors are not propagated to the proteins' surfaces, preserving the protein-protein interactions with their three common partners, FakA, PlsX, and PlsY. Using cellular thermal shift analyses, we found that FakB1 binds FA in vivo, whereas a significant proportion of FakB2 does not. Incorporation of exogenous FA into phospholipid in ?fakB1 and ?fakB2 S. aureus knockout strains revealed that FakB1 does not efficiently activate unsaturated FAs. FakB2 preferred unsaturated FAs, but also allowed the incorporation of saturated FAs. These results are consistent with a model in which FakB1 primarily functions in the recycling of the saturated FAs produced by S. aureus metabolism, whereas FakB2 activates host-derived oleate, which S. aureus does not produce but is abundant at infection sites.

| S-EPMC6322867 | biostudies-literature

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