The acyl-enzyme mechanism of beta-lactamase action. The evidence for class C Beta-lactamases.
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ABSTRACT:
SUBMITTER: Knott-Hunziker V
PROVIDER: S-EPMC1153862 | biostudies-other | 1982 Nov
REPOSITORIES: biostudies-other
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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: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
Project description:Optimization by energy minimization of stable complexes occurring along the pathway of hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase has highlighted a proton shuttle that may explain the catalytic mechanism of the beta-lactamases of class A. Five residues, S70, S130, N132, T235 and A237, are involved in ligand binding. The gamma-OH group of T235 and, in the case of benzylpenicillin, the gamma-OH group of S130 interact with the carboxylate group, on one side of the ligand molecule. The side-chain NH2 group of N132 and the carbonyl backbone of A237 interact with the exocyclic CONH amide bond, on the other side of the ligand. The backbone NH groups of S70 and A237 polarize the carbonyl group of the scissile beta-lactam amide bond. Four residues, S70, K73, S130 and E166, and two water molecules, W1 and W2, perform hydrolysis of the bound beta-lactam compound. E166, via W1, abstracts the proton from the gamma-OH group of S70. While losing its proton, the O-gamma atom of S70 attacks the carbonyl carbon atom of the beta-lactam ring and, concomitantly, the proton is delivered back to the adjacent nitrogen atom via W2, K73 and S130, thus achieving formation of the acyl-enzyme. Subsequently, E166 abstracts a proton from W1. While losing its proton, W1 attacks the carbonyl carbon atom of the S70 ester-linked acyl-enzyme and, concomitantly, re-entry of a water molecule W'1 replacing W1 allows E166 to deliver the proton back to the same carbonyl carbon atom, thus achieving hydrolysis of the beta-lactam compound and enzyme recovery. The model well explains the differences found in the kcat. values for hydrolysis of benzylpenicillin and cephalosporin C by the Streptomyces albus G beta-lactamase. It also explains the effects caused by site-directed mutagenesis of the Bacillus cereus beta-lactamase I [Gibson, Christensen & Waley (1990) Biochem J. 272, 613-619].
| S-EPMC1151568 | biostudies-other
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
Project description:Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with beta-lactam and quinolone antibiotics. The isolate produced an inducible class A beta-lactamase of pI 8.6, named Sed-1, which was purified. Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin. The corresponding gene, bla(Sed-1), was cloned and sequenced. Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded beta-lactamases from Citrobacter koseri (formerly C. diversus) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), and Proteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1. A gene coding for a LysR transcriptional regulator was found upstream from bla(Sed-1). This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal beta-lactamase from C. koseri and 63 and 50% identity with the AmpR sequences of P. vulgaris and Enterobacter cloacae, respectively. By using DNA-DNA hybridization, a bla(Sed-1)-like gene was identified in two reference strains, C. sedlakii (CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C. koseri studied. Two DNA fragments were amplified and sequenced from the reference strains of C. sedlakii CIP-105037 and C. rodentium CIP-104675 using two primers specific for bla(Sed-1). They shared 98 and 80% identity with bla(Sed-1), respectively, confirming the diversity of the chromosomally encoded class A beta-lactamases found in Citrobacter.
| S-EPMC90644 | biostudies-literature
Project description:The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in Acinetobacter baumannii, including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant A. baumannii strains constitutes a serious global threat. Carbapenems have been successfully utilized as last-resort antibiotics for the treatment of multidrug-resistant A. baumannii infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work was to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in A. baumannii The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against A. baumannii class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor that may have a significant role in eradicating infections caused by A. baumannii isolates carrying CHDLs.
| S-EPMC5655052 | biostudies-literature
Project description:β-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αβ domains conjugated by an interdomain loop and can effectively bind and inactivate class A β-lactamases, which are responsible for resistance of bacteria to β-lactam antibiotics. The varied ability of BLIP to bind different β-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A β-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A β-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.
| S-EPMC8363833 | biostudies-literature
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
Project description:Class C β-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs (64SXSK, 150YXN, 315KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera Legionella, Bradyrhizobium, and Parachlamydia) has raised questions about the possible existence of a C2 subclass of β-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC β-lactamases (ESACs), the genetic modifications observed in vivo and in vitro (point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet 150YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304-306), and in Hafnia alvei and H. paralvei (deletion of residues 289-290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to β-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes (ampR, ampD, ampG, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.
| S-EPMC9491196 | biostudies-literature
Project description:The reactions between single crystals of the SHV-1 beta-lactamase enzyme and the carbapenems, meropenem, imipenem, and ertapenem, have been studied by Raman microscopy. Aided by quantum mechanical calculations, major populations of two acyl-enzyme species, a labile Delta (2)-pyrroline and a more tightly bound Delta (1)-pyrroline, have been identified for all three compounds. These isomers differ only in the position of the double bond about the carbapenem nucleus. This discovery is consonant with X-ray crystallographic findings that also identified two populations for meropenem bound in SHV-1: one with the acyl CO group in the oxyanion hole and the second with the acyl group rotated 180 degrees compared to its expected position [Nukaga, M., Bethel, C. R., Thomson, J. M., Hujer, A. M., Distler, A. M., Anderson, V. E., Knox, J. R., and Bonomo, R. A. (2008) J. Am. Chem. Soc. (in press)]. When crystals of the Delta (1)- and Delta (2)-containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Delta (2) species was observed by kinetic Raman experiments. However, no change in the Delta (1) population was observed over 1 h, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Delta (1) species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Delta (2) species corresponds to the form with the carbonyl inside the oxyanion hole. Soak-in and soak-out Raman experiments also demonstrated that tautomeric exchange between the Delta (1) and Delta (2) forms does not occur on the crystalline enzyme. When meropenem or ertapenem was reacted with SHV-1 in solution, the Raman difference spectra demonstrated that only a major population corresponding to the Delta (1) acyl-enzyme could be detected. The 1003 cm (-1) mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal Raman intensity standard, and the ratio of its intensity to that of the 1600 cm (-1) feature of Delta (1) provides an estimate of the relative populations of Delta (1). In solution, I 1600/ I 1003 equals 2, and in the crystal, I 1600 /I 1003 equals 1. This is strong evidence that the Delta (1) and Delta (2) acyl-enzymes in the crystal are present in approximately equal amounts, in agreement with the X-ray data. However, in solution there are twice as many Delta (1) species per Phe group, and this represents approximately 100% of the active sites, which is consistent with the observed inhibition of the enzyme's activity.
| S-EPMC2656688 | biostudies-literature