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The importance of the negative charge of beta-lactam compounds in the interactions with active-site serine DD-peptidases and beta-lactamases.

ABSTRACT: The interaction between various penicillins and cephalosporins the carboxylate group of which at C-3 or C-4 had been esterified or amidated and different penicillin-recognizing enzymes was studied. In general, our findings reinforced the common assumption that an anionic group at that position is necessary for the effective acylation of these enzymes. However, the relative activities of the modified beta-lactams as inactivators of the Streptomyces R61 DD-peptidase or as substrates of the Bacillus licheniformis, Streptomyces albus G and Enterobacter cloacae beta-lactamases did not fit a general scheme in which the intrinsic electronic and geometric properties of the beta-lactam compounds would be sufficient to explain their substrate or inactivator properties towards the various types of enzymes investigated.

SUBMITTER: Varetto L

PROVIDER: S-EPMC1151417 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Interactions of "bora-penicilloates" with serine ?-lactamases and DD-peptidases.

Project description:Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial ?-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two ?-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in ?-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C ?-lactamases but, very unexpectedly, not inhibitors of class A ?-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of ?-lactam antibiotics, where deacylation of ?-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

| S-EPMC4204886 | biostudies-literature

Evidence for an oxyanion hole in serine beta-lactamases and DD-peptidases.

Project description:A thionocephalosporin is shown to be a much poorer substrate of representative serine beta-lactamases of class A (RTEM-2) and class C (Enterobacter cloacae P99) and a much poorer inhibitor of the Streptomyces R61 DD-peptidase than is the analogous oxo beta-lactam. These results provide kinetic evidence for the existence of a catalytic oxyanion hole in these enzymes.

| S-EPMC1135462 | biostudies-other

Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-?-hydroxyglycine ester catalyzed by serine ?-lactamases and DD-peptidases.

Project description:The ?-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-?-hydroxyglycinate (5) is a quite effective substrate of serine ?-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC ?-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 ?-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since ?-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine ?-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the ?-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 ?-lactamase active site suggests that the ?-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both ?-hydroxy and ?-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the ?-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

| S-EPMC3684201 | biostudies-literature

Chromogenic depsipeptide substrates for beta-lactamases and penicillin-sensitive DD-peptidases.

Project description:Various ester and thioester derivatives of hippuric acid have been prepared which were substrates of both beta-lactamases and DD-peptidases. The thioesters were more rapidly hydrolysed by nearly all the enzymes. Surprisingly, the enzymes acted rather efficiently on substrates which did not contain any chiral centre.

| S-EPMC1131754 | biostudies-other

Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β-Lactamases.

Project description:Bacterial resistance to standard (i.e., β-lactam-based) antibiotics has become a global pandemic. Simultaneously, research into the underlying causes of resistance has slowed substantially, although its importance is universally recognized. Key to unraveling critical details is characterization of the noncovalent interactions that govern binding and specificity (DD-peptidases, antibiotic targets, versus β-lactamases, the evolutionarily derived enzymes that play a major role in resistance) and ultimately resistance as a whole. Herein, we describe a detailed investigation that elicits new chemical insights into these underlying intermolecular interactions. Benzylpenicillin and a novel β-lactam peptidomimetic complexed to the Stremptomyces R61 peptidase are examined using an arsenal of computational techniques: MD simulations, QM/MM calculations, charge perturbation analysis, QM/MM orbital analysis, bioinformatics, flexible receptor/flexible ligand docking, and computational ADME predictions. Several key molecular level interactions are identified that not only shed light onto fundamental resistance mechanisms, but also offer explanations for observed specificity. Specifically, an extended π-π network is elucidated that suggests antibacterial resistance has evolved, in part, due to stabilizing aromatic interactions. Additionally, interactions between the protein and peptidomimetic substrate are identified and characterized. Of particular interest is a water-mediated salt bridge between Asp217 and the positively charged N-terminus of the peptidomimetic, revealing an interaction that may significantly contribute to β-lactam specificity. Finally, interaction information is used to suggest modifications to current β-lactam compounds that should both improve binding and specificity in DD-peptidases and their physiochemical properties.

| S-EPMC3985439 | biostudies-literature

Breakdown of the stereospecificity of DD-peptidases and beta-lactamases with thiolester substrates.

Project description:With peptide analogues of their natural substrates (the glycopeptide units of nascent peptidoglycan), the DD-peptidases exhibit a strict preference for D-Ala-D-Xaa C-termini. Gly is tolerated as the C-terminal residue, but with a significantly decreased activity. These enzymes were also known to hydrolyse various ester and thiolester analogues of their natural substrates. Some thiolesters with a C-terminal leaving group that exhibited L stereochemistry were significantly hydrolysed by some of the enzymes, particularly the Actinomadura R39 DD-peptidase, but the strict specificity for a D residue in the penultimate position was fully retained. These esters and thiolesters also behave as substrates for beta-lactamases. In this case, thiolesters exhibiting L stereochemistry in the ultimate position could also be hydrolysed, mainly by the class-C and class-D enzymes. However, more surprisingly, the class-C Enterobacter cloacae P99 beta-lactamase also hydrolysed thiolesters containing an L residue in the penultimate position, sometimes with a higher efficiency than the D isomer.

| S-EPMC1135750 | biostudies-other

Interaction between non-classical beta-lactam compounds and the Zn2+-containing G and serine R61 and R39 D-alanyl-D-alanine peptidases.

Project description:Streptomyces albus G secretes a Zn2+-containing D-alanyl-D-alanine peptidase. Streptomyces R61 and Actinomadura R39 secrete D-alanyl-D-alanine-cleaving serine peptidases. The effect of non-classical beta-lactam antibiotics on these three model enzymes has been studied. Mecillinam, cefoxitin, quinacillin, quinacillin sulphone, clavulanate and N-formimidoylthienamycin have no effect on the Zn2+-containing enzyme. 6-Amino-penicillanic acid slowly inactivates this enzyme and 7-aminocephalosporanic acid behaves as a reversible inhibitor. Cefoxitin and N-formimidoylthienamycin are potent anti-bacterial agents; they effectively inactivate the serine R39 enzyme and, to a lesser extent, the serine R61 enzyme. All the other beta-lactam compounds tested, including mecillinam, are slow inactivators of these serine enzymes. The intermediates formed between 6-aminopenicillanic acid and the R61 and R39 enzymes are long- and short-lived respectively, whereas those formed between 7-aminocephalosporanic acid and the same R61 and R39 enzymes are short- and long-lived respectively. Breakdown of the short-lived intermediates thus obtained gives rise to several ninhydrin-positive degradation products. The intermediates formed between clavulanate and the serine enzymes are long-lived. With the R39 enzyme, the inactivated complex formed in a first step undergoes subsequent monomolecular rearrangement to give rise to a second species exhibiting a high absorbance at 273 nm.

| S-EPMC1163342 | biostudies-other

A survey of the kinetic parameters of class C beta-lactamases. Cephalosporins and other beta-lactam compounds.

Project description:Various cephalosporins, cefoxitin, moxalactam, imipenem and aztreonam were studied as substrates of six class C beta-lactamases. Nitrocefin, cephaloridine, cefazolin, cephalothin and cephalexin were good substrates, with kcat. values ranging from 27 to 5000 s-1. Cefuroxime, cefotaxime and cefoxitin exhibited low kcat. values (0.010-1.7 s-1) and low Km values, which suggested a rate-limiting deacylation. Imipenem and aztreonam were even poorer substrates (kcat. 2 x 10(-4)-3 x 10(-2) s-1) and, in the presence of a reporter substrate, behaved as transient inactivators. With moxalactam, biphasic kinetics were observed, indicating a possible rearrangement of the acyl-enzyme.

| S-EPMC1135199 | biostudies-other

BADAN-conjugated β-lactamases as biosensors for β-lactam antibiotic detection.

Project description:β-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated β-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for β-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP β-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to β-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four β-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 μM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated β-lactamases as biosensing devices for β-lactam antibiotics.

| S-EPMC7598492 | biostudies-literature

Interactions between non-classical beta-lactam compounds and the beta-lactamases of Actinomadura R39 and Streptomyces albus G.

Project description:6-Aminopenicillanic acid, 7-aminocephalosporanic acid, mecillinam and quinacillin have varying substrate activities for both the R39 beta-lactamase (excreted by Actinomadura R39) and the G beta-lactamase (excreted by Streptomyces albus G). Cefoxitin and quinacillin sulphone are not recognized by the G beta-lactamase and are weak inactivators of the R39 beta-lactamase. N-Formimidoylthienamycin is a poor substrate for the G beta-lactamase and a potent inactivator of the R39 beta-lactamase. The high value of the bimolecular rate constant for enzyme inactivation is mainly due to a very low dissociation constant (1 microM). Clavulanate is an inactivator of both G and R39 beta-lactamases. The reaction with this latter enzyme is a branched pathway where normal turnover and permanent enzyme inactivation occur concomitantly. Between 28 and 43 molecules of clavulanate are hydrolysed before one of them has the opportunity to inactivate one molecule of enzyme.

| S-EPMC1163343 | biostudies-other

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