Molecular dynamics simulations of class C beta-lactamase from Citrobacter freundii: insights into the base catalyst for acylation

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.     

Neutron Diffraction Studies of a Class A β-Lactamase Toho-1 E166A/R274N/R276N Triple Mutant

β-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the β-lactam antibiotics is the production of β-lactamase enzymes that inactivate β-lactams by rapidly hydrolyzing the amide group of the β-lactam ring. Specifically, the class A extended-spectrum β-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a β-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.     

The roles of residues Tyr150, Glu272, and His314 in class C β-lactamases

Serine β-lactamases contribute widely to the β-lactam resistance phenomena. Unfortunately, the intimate details of their catalytic mechanism remain elusive and subject to some controversy even though many “natural” and “artificial” mutants of these different enzymes have been isolated. This paper is essentially focused on class C β-lactamases, which contain a Tyr (Tyr150) as the first residue of the second conserved element, in contrast to their class A counterparts, in which a Ser is found in the corresponding position. We have modified this Tyr residue by site-directed mutagenesis. On the basis of the three-dimensional structure of the Enterobacter cloacae P99 enzyme, it seemed that residues Glu272 and His314 might also be important. They were similarly substituted. The modified enzymes were isolated and their catalytic properties determined. Our results indicated that His314 was not required for catalysis and that Glu272 did not play an important role in acylation but was involved to a small extent in the deacylation process. Conversely, Tyr150 was confirmed to be central for catalysis, at least with the best substrates. On the basis of a comparison of data obtained for several class C enzyme mutants and in agreement with recent structural data, we propose that the phenolate anion of Tyr150, in conjunction with the alkyl ammonium of Lys315, acts as the general base responsible for the activation of the active-site Ser64 during the acylation step and for the subsequent activation of a water molecule in the deacylation process. The evolution of the important superfamily of penicillin-recognizing enzymes is further discussed in the light of this proposed mechanism. © 1996 Wiley-Liss, Inc.     

Structural basis for the β‐lactamase activity of EstU1, a family VIII carboxylesterase

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β‐lactam antibiotics as well as the ester bond of p‐nitrophenyl esters. EstU1 assumes a β‐lactamase‐like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β‐lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β‐lactam antibiotics. This result explains the weak β‐lactamase activity of EstU1 compared with class C β‐lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum. Proteins 2013; 81:2045–2051. © 2013 Wiley Periodicals, Inc.     

Serotype distribution and β-lactam resistance in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> isolated from patients with respiratory infections in Korea

Haemophilus influenzae is a frequent causative bacterial pathogen of respiratory tract infections. Resistance to β-lactam antibiotics has been a significant clinical problem in treatment for H. influenzae respiratory infections. This study describes the serotype, antibiotic resistance and distribution of TEM-1 or ROB-1 β-lactamase in H. influenzae isolates from local private hospitals from 2002 to 2004. Among the 100 H. influenzae respiratory isolates, only 7% were identified as serotypes a, b, e, and f, with the remaining 93% being nontypeable. Resistance to ampicillin, cefaclor, and tetracycline was 57%, 46%, and 16%, respectively. All strains were susceptible to azithromycin and ciprofloxacin, whereas amoxicillin/clavulanate, cefotaxime, and imipenem exhibited reduced susceptibilities of 99%, 99%, and 91%, respectively. All 57 ampicillinresistant strains (minimum inhibitory concentration, MIC≥4 μg/ml) were β-lactamase-positive and possessed the TEM-1 type β-lactamase. One β-lactamase-positive amoxicillin/clavulanate-resistant isolate that was resistant to ampicillin (MIC>128 μg/ml) had the TEM-1 type β-lactamase and not susceptible to cefaclor and cefotaxime. Analysis of penicillin binding protein 3 revealed six residues (Asp-350, Met-377, Ala-502, Asn-526, Val-547, and Asn-569) that were substituted by Asn, Ile, Val, Lys, Ile, and Ser, respectively.     

Molecular Insights on the Release of Avibactam from the Acyl-Enzyme Complex

Avibactam is a non-β-lactam β-lactamase inhibitor for treating complicated urinary tract and respiratory infections caused by multidrug-resistant bacterial pathogens, a serious public health threat. Despite its importance, the release mechanism of avibactam from the enzyme-inhibitor complex has been scarcely studied from first principles, considering the total protein environment. This information at the molecular level is essential for the rational design of new antibiotics and inhibitors. In this article, we addressed the release of avibactam from the complex CTX-M-15 by means of molecular dynamics simulations and quantum mechanics/molecular mechanics calculations. This study provides molecular information not available earlier, including exploration of the potential energy surfaces, characterization of the observed intermediate, and their critical points, as well. Our results show that unlike that observed in the acylation reaction, the residues Glu166 and Lys73 would be in their neutral forms. Release of avibactam follows a stepwise mechanism in which the first stage corresponds to the formation of a tetrahedral intermediate, whereas the second stage corresponds to the cleavage of the Ser70-C7 bond, mediated by Lys73, either directly or through Ser130.     

Susceptibility of Gram-negative bacteria to β-lactam antibiotics and rapid characterization of β-lactamase activity

Various species of Gram-negative bacteria were tested for susceptibility to β-lactam antibiotics and for production of β-lactamase. The rapid cephalosporin 87/312 visual test was more sensitive than either the acidometric or the iodometric test; the iodometric test was least sensitive. Characteristic β-lactamase hydrolysis product patterns were obtained by scanning mixtures of β-lactamase-producing bacteria and cephalosporin substrates. β-Lactamase could not be detected on bacterial cells by fluorescent antibody techniques. The presence of β-lactamase can be correlated with minimal inhibitory concentrations of β-lactam antibiotics only in certain Gram-negative bacilli.     

Re‐examining the role of Lys67 in class C β‐lactamase catalysis

Lys67 is essential for the hydrolysis reaction mediated by class C β‐lactamases. Its exact catalytic role lies at the center of several different proposed reaction mechanisms, particularly for the deacylation step, and has been intensely debated. Whereas a conjugate base hypothesis postulates that a neutral Lys67 and Tyr150 act together to deprotonate the deacylating water, previous experiments on the K67R mutants of class C β‐lactamases suggested that the role of Lys67 in deacylation is mainly electrostatic, with only a 2‐ to 3‐fold decrease in the rate of the mutant vs the wild type enzyme. Using the Class C β‐lactamase AmpC, we have reinvestigated the activity of this K67R mutant enzyme, using biochemical and structural studies. Both the rates of acylation and deacylation were affected in the AmpC K67R mutant, with a 61‐fold decrease in k_cat, the deacylation rate. We have determined the structure of the K67R mutant by X‐ray crystallography both in apo and transition state‐analog complexed forms, and observed only minimal conformational changes in the catalytic residues relative to the wild type. These results suggest that the arginine side chain is unable to play the same catalytic role as Lys67 in either the acylation or deacylation reactions catalyzed by AmpC. Therefore, the activity of this mutant can not be used to discredit the conjugate base hypothesis as previously concluded, although the reaction catalyzed by the K67R mutant itself likely proceeds by an alternative mechanism. Indeed, a manifold of mechanisms may contribute to hydrolysis in class C β‐lactamases, depending on the enzyme (wt or mutant) and the substrate, explaining why different mutants and substrates seem to support different pathways. For the WT enzyme itself, the conjugate base mechanism may be well favored.     

Occurrence and transferability of β-lactam resistance inEnterobacteriaceae isolated inChildren's University Hospital in Bratislava

Occurrence and transferability of β-lactam resistance in 30 multi-resistantEscherichia coli, Klebsiella spp.,Enterobacter spp.,Pantoea agglomerans, Citrobacter freundii andSerratia marcescens strains isolated from children between 0 and 3 years of age is presented. The strains were resistant to ampicillin (30), cefoxitin (22), cefotaxime (30), ceftriaxone (30), ceftazidime (30) and aztreonam (28), but susceptible to cefepime (30) and imipenem (26). Twenty-eight of 30 isolates possessed a transferable resistance confirmed by conjugation and isolation of 79–89-kb plasmids. The β-lactam resistance was due to production of β-lactamases and ceftazidime proved to be stronger β-lactamase inductor than ceftriaxone. Twenty-five clinical isolates expressed transferable extended spectrum β-lactamases, and chromosomally encoded AmpC β-lactamase.     

A new method of colorimetric assay of β-lactamase suitable for estimation of β-lactamase inhibition in crude microbial culture filtrates

A simple procedure is described for the rapid assay of β-lactamases suitable for use in the quantitation of β-lactamase inhibition in crude culture filtrates of soil microbes. The proposed method is based on measuring the blue reaction product (λ_max 750 nm) formed during reduction of phosphomolybdic acid with reducing products generated from β-lactam hydrolysis and is not influenced significantly by the metabolites of microbial culture filtrates which often interfere with conventional β-lactamase assay techniques.     

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

Background  

β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.     

Type I β-lactamases ofEnterobacter cloacae and resistance to β-lactam antibiotics

The interaction of type-I β-lactamases fromEnterobacter cloacae with diverse β-lactam compounds was examined. The ability of penicillin and cefoxitin to induce β-lactamase production in this strain was assessed. The effect of β-lactamase inhibitors was measured on β-lactamase extracts and on intact cells.E. cloacae 78 strain is a stably derepressed mutant showing limited susceptibility to a number of antibiotics except imipenem. Imipenem would therefore be the appropiate choice for therapy of infections caused by stably derepressed mutants ofEnterobacter sp. producing type-I β-lactamases.     

Molecular modeling of Henry-Michaelis and acyl-enzyme complexes between imipenem and Enterobacter cloacae P99 beta-lactamase

We report a molecular-mechanics (AMBER*) study on the Henry-Michaelis complex and the corresponding acyl-enzyme adduct formed between imipenem (1), a transient inhibitor of beta-lactamases, and Enterobacter cloacae P99, a class C-beta-lactamase. We have examined the influence of the structural configuration of the functional groups in the substrate on their three-dimensional (3D) arrangement at the active site, which was compared with those adopted by typical penicillins and cephalosporins. Our results confirm that the carboxy group of the antibiotic plays a prominent role in the binding of the substrate to the active site, and that it activates Ser64 through interaction with the phenolic OH group of Tyr150. The binding of imipenem to E. cloacae P99 increases the distance between Tyr150 and Ser64 due to the presence of a hydrophobic Me group in the (R)-1-hydroxyethyl substituent at C(6). This, together with the 3D arrangement of its carboxy group, leads to an interaction with the active site in a manner that hinders H+ exchange between the nucleophile in Ser64 and its basic activator, the phenolic group of Tyr150.     

Structural studies of the mechanism for biosensing antibiotics in a fluorescein-labeled <Emphasis Type="Italic">β</Emphasis>-lactamase

Background  

β-lactamase conjugated with environment-sensitive fluorescein molecule to residue 166 on the Ω-loop near its catalytic site is a highly effective biosensor for β-lactam antibiotics. Yet the molecular mechanism of such fluorescence-based biosensing is not well understood.     

Studies on amino acid replacement and inhibitory activity of a β-lactamase inhibitory peptide

An SHV β-lactamase gene was amplified from a β-lactam resistant Klebsiella pneumoniae K-71 genomic DNA. After expression and purification, we demonstrated that peptide P1 could inhibit the hydrolysis activity of both TEM-1 and SHV β-lactamase in vitro. Three mutations were introduced into P1 in which the first residue S was replaced by F, the 18th residue V was mutated to Y, and the 15th residue Y was substituted with A, C, G, and R to obtain the mutants of P1-A, P1- C, P1-G, and P1-R, respectively. The mutant peptides were purified and their inhibitory constants against TEM-1 and SHV β-lactamase were determined. All these β-lactamase inhibitory peptides could inhibit the activity of both β-lactamases, while the mutant peptides showed stronger inhibitory activities against TEM-1 β-lactamase than against SHV β-lactamase. Inhibition data suggested that P1-A improved the β-lactamase inhibitory activity by over 3-fold compare to P1. When P1-A was incubated with K. pneumoniae K-71 in Luria-Bertani medium containing ampicillin, it showed a much stronger growth of inhibition ratio over P1. This study gives us a good candidate for development of novel β-lactamase inhibitors.     

Clavulanic acid biosynthesis and genetic manipulation for its overproduction

Clavulanic acid, a β-lactamase inhibitor, is used together with β-lactam antibiotics to create drug mixtures possessing potent antimicrobial activity. In view of the clinical and industrial importance of clavulanic acid, identification of the clavulanic acid biosynthetic pathway and the associated gene cluster(s) in the main producer species, Streptomyces clavuligerus, has been an intriguing research question. Clavulanic acid biosynthesis was revealed to involve an interesting mechanism common to all of the clavam metabolites produced by the organism, but different from that of other β-lactam compounds. Gene clusters involved in clavulanic acid biosynthesis in S. clavuligerus occupy large regions of nucleotide sequence in three loci of its genome. In this review, clavulanic acid biosynthesis and the associated gene clusters are discussed, and clavulanic acid improvement through genetic manipulation is explained.     

pKa calculations for class A beta-lactamases: methodological and mechanistic implications.

Beta-lactamases are responsible for resistance to penicillins and related beta-lactam compounds. Despite numerous studies, the identity of the general base involved in the acylation step is still unclear. It has been proposed, on the basis of a previous pKa calculation and analysis of structural data, that the unprotonated Lys73 in the active site could act as the general base. Using a continuum electrostatic model with an improved treatment of the multiple titration site problem, we calculated the pKa values of all titratable residues in the substrate-free TEM-1 and Bacillus licheniformis class A beta-lactamases. The pKa of Lys73 in both enzymes was computed to be above 10, in good agreement with recent experimental data on the TEM-1 beta-lactamase, but inconsistent with the proposal that Lys73 acts as the general base. Even when the closest titratable residue, Glu166, is mutated to a neutral residue, the predicted downward shift of the pKa of Lys73 shows that it is unlikely to act as a proton abstractor in either enzyme. These results support a mechanism in which the proton of the active Ser70 is transferred to the carboxylate group of Glu166.     

Structure of the extended-spectrum class C beta-lactamase of Enterobacter cloacae GC1, a natural mutant with a tandem tripeptide insertion.

A class C beta-lactamase from a clinical isolate of Enterobacter cloacae strain GC1 with improved hydrolytic activity for oxyimino beta-lactam antibiotics has been analyzed by X-ray crystallography to 1.8 A resolution. Relative to the wild-type P99 beta-lactamase, this natural mutant contains a highly unique tandem repeat Ala211-Val212-Arg213 [Nugaka et al. (1995) J. Biol. Chem. 270, 5729-5735]. The 39.4 kDa chromosomal beta-lactamase crystallizes from poly(ethylene glycol) 8000 in potassium phosphate in space group P2(1)2(1)2 with cell dimensions a = 78.0 A, b = 69.5 A, and c = 63.1 A. The crystal structure was solved by the molecular replacement method, and the model has been refined to an R-factor of 0.20 for all nonzero data from 8 to 1.8 A. Deviations of model bonds and angles from ideal values are 0.008 A and 1.4 degrees, respectively. Overlay of alpha-carbon atoms in the GC1 and P99 beta-lactamases results in an rms deviation of 0.6 A. Largest deviations occur in a loop containing Gln120 and in the Omega loop region (200-218) where the three residues 213-215 are disordered. Possibly as a result of this disorder, the width of the opening to the substrate binding cavity, as measured from the 318-324 beta-strand to two loops containing Gln120 and Tyr150 on the other side, is 0.6-1.4 A wider than in P99. It is suggested that conformational flexibility in the expanded Omega loop, and its influence on adjacent protein structure, may facilitate hydrolysis of oxyimino beta-lactams by making the acyl intermediate more open to attack by water. Nevertheless, backbone atoms in core catalytic site residues Ser64, Lys67, Tyr150, Asn152, Lys318, and Ser321 deviate only 0.4 A (rmsd) from atoms in P99. A rotation of a potential catalytic base, Tyr150, relative to P99 at pH 8, is consistent with the requirement for a lower than normal pK(a) for this residue.