A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.     

Kinetics and regulation of beta-lactamase formation in methylotrophic bacterium

The regulation and kinetics of formation of an inducible, cell-bound oxacillin-hydrolyzing beta-lactamase (M-OXA) by a methanol-grown Pseudomonas strain were investigated in batch, chemostat, and two-stage continuous cultures. The extent to which enzyme production occurs declines at increased growth rates, and the rate of M-OXA beta-lactamase production follows a kinetic pattern that is partially growth independent and partially inversely growth linked. Growth and enzyme formation are regulated differently by medium constituents. The initial steps of M-OXA beta-lactamase synthesis takes place during the exponential growth phase, and active enzyme is produced by induced nonproliferating cells, probably through a turnover mechanism.     

Beta-lactamases from Yersinia enterocolitica.

Two beta-lactamases, A and B, have been shown to be present in a strain of Yersinia enterocolitica (w222). Beta-Lactamase A hydrolyses a variety of penicillins and cephalosporins. This enzyme is sensitive to thiol reagents, is only partially inhibited by 0-1 mM-cloxacillin and has a molecular weight of approximatley 20,000.beta-Lactamase B shows strong cephalosporinase activity but does not hydrolyse some of the penicillins. It is more resistant than beta-lactamase A to thiol reagents, is completely inhibited by 0-1 mM-cloxacillin and has a molecular weight of about 34,000. With cephaloridine as a substrate, which is readily hydrolysed by both enzymes, about 85% of the total activity of a cell extract is due to beta-lactamase A and 15% to B. Addition of 6-aminopenicillanic acid to the culture during growth results in a 2-to4-fold selective increase in the amount of beta-lactamase B. Two beta-lactamases similar to enzymes A and B have been found in five other strains of Y. enterocolitica. In contrast, only one beta-lactamase, similar to enzyme B, has been detected in a different strain of Y. enterocolitica (H66), which is abnormal in that it is sensitive to ampicillin. Addition of 6-aminopenicillanic acid to cultures of this strain results in an 8-to 10-fold increase in beta-lactamase production.     

Beta-lactamase of Lysobacter enzymogenes: induction, purification and characterization

Lysobacter enzymogenes produces an inducible beta-lactamase and induction with 100 micrograms ampicillin ml-1 resulted in an increase of more than 100-fold in enzyme activity. Various other beta-lactam antibiotics also served as effective inducers. The enzyme was obtained from cells by osmotic shocking to release periplasmic components and it was purified primarily by ion-exchange chromatography and PAGE. The beta-lactamase consists of one polypeptide with a molecular mass of about 28 kDa and an isoelectric point greater than 9.6. It is strongly inhibited by p-chloromercuribenzoate and clavulanic acid but not by EDTA. The enzyme readily hydrolyses several penicillins and cephalosporins, but not oxacillin or cloxacillin. The enzyme therefore belongs to group 2b of the bacterial beta-lactamases.     

Calorimetric analysis of cephalosporins using an immobilized TEM-1 beta-lactamase on Ni2+ chelating sepharose fast flow

Two beta-lactamases, penicillinase type I from Bacillus cereus and TEM-1 beta-lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of beta-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 beta-lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone.     

Replacement of lysine 234 affects transition state stabilization in the active site of beta-lactamase TEM1

Lysine 234 is a residue highly conserved in all beta-lactamases, except in the carbenicillin-hydrolyzing enzymes, in which it is replaced by an arginine. Informational suppression has been used to create amino acid substitutions at this position in the broad spectrum Escherichia coli beta-lactamase TEM-1, in order to elucidate the role of this residue which lies on the wall at the closed end of the active site cavity. The mutants K234R and K234T were constructed and their kinetic constants measured. Replacement of lysine 234 by arginine yields an enzyme with similar activity toward cephalosporins and most penicillins, except toward the carboxypenicillins for which the presence of the guanidine group enhances the transition state binding. The removal of the basic group in the mutant K234T yields a protein variant which retains a low activity toward penicillins, but losts drastically its ability to hydrolyze cephalosporins. Moreover, these two mutations largely decreased the affinity of the enzyme for penicillins (10-fold for K234R and 50-fold for K234T). This can be correlated with the disruption of the predicted electrostatic binding between the C3 carboxylic group of penicillins and the amine function of the lysine. Therefore, lysine 234 in the E. coli beta-lactamase TEM-1 is involved both in the initial recognition of the substrate and in transition state stabilization.     

Comparison of the Substrate Specificities of the β-Lactamases from Klebsiella aerogenes 1082E and Enterobacter cloacae P99

A potent beta-lactamase (EC 3.5.2.6) produced by a strain of Klebsiella aerogenes (K. pneumoniae), 1082E, isolated from a hospital patient, has been examined. Its properties were different from those of most gram-negative beta-lactamases previously reported. The enzyme has been partly purified, and its activity against a range of substrates has been compared with that of the enzyme from Enterobacter cloacae (Aerobacter cloacae) P99. The K. aerogenes enzyme, although predominantly a penicillinase, had a wide range of specificity. In addition to hydrolyzing the cephalosporins, it attacked the normally beta-lactamaseresistant compounds methicillin and cloxacillin as well as cephalosporin analogues with the same acyl substituents. The results obtained with the E. cloacae enzyme confirmed its cephalosporinase activity and showed that, unlike the enzyme from K. aerogenes, it was relatively inactive against the penicillins.     

A new family of cyanobacterial penicillin-binding proteins. A missing link in the evolution of class A beta-lactamases

It is largely accepted that serine beta-lactamases evolved from some ancestral DD-peptidases involved in the biosynthesis and maintenance of the bacterial peptidoglycan. DD-peptidases are also called penicillin-binding proteins (PBPs), since they form stable acyl-enzymes with beta-lactam antibiotics, such as penicillins. On the other hand, beta-lactamases react similarly with these antibiotics, but the acyl-enzymes are unstable and rapidly hydrolyzed. Besides, all known PBPs and beta-lactamases share very low sequence similarities, thus rendering it difficult to understand how a PBP could evolve into a beta-lactamase. In this study, we identified a new family of cyanobacterial PBPs featuring the highest sequence similarity with the most widespread class A beta-lactamases. Interestingly, the Omega-loop, which, in the beta-lactamases, carries an essential glutamate involved in the deacylation process, is six amino acids shorter and does not contain any glutamate residue. From this new family of proteins, we characterized PBP-A from Thermosynechococcus elongatus and discovered hydrolytic activity with synthetic thiolesters that are usually good substrates of DD-peptidases. Penicillin degradation pathways as well as acylation and deacylation rates are characteristic of PBPs. In a first attempt to generate beta-lactamase activity, a 90-fold increase in deacylation rate was obtained by introducing a glutamate in the shorter Omega-loop.     

Purification and properties of a constitutive beta-lactamase from Pseudomonas aeruginosa strain Dalgleish.

1. The beta-lactamase (penicillin amido-beta-lactamhydrolase EC 3.5.2.6) appeared to be periplasmic rather than truly intracellular, since it was released by freeze-thawing without gross morphological changes in the cell. 2. The partially purified enzyme had pI between 5.0 and 5.5, mol. wt 32 000 and a broad pH vs activity profile with a maximum at pH 8. 3. The cephalosporins tested were hydrolysed less rapidly than most of the penicillins, and the Km values for penicillins were lower than for cephalosporins. However cloxacillin was hydrolysed very slowly although it was strongly bound. The substrate-induced inactivation common to many beta-lactamases was particularly marked with cephaloridine and cloxacillinmthe cloxacillin-induced inactivation was shown to be reversible.     

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.     

TEM-1 beta-lactamase as a scaffold for protein recognition and assay

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 beta-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to beta-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that beta-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.     

Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.     

beta-lactamase from Streptomyces cacaoi. Purification and properties

A beta-lactamase was purified to an apparently homogeneous state from Streptomyces cacaoi. The molecular weight calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis was 34,000. pI was 4.7 and the optimal pH was 6.5. The optimum temperature was found to be between 40 degrees C and 45 degrees C, but the enzyme lost activity above 50 degrees C. N-Bromosuccinimide was the strongest inhibitor among the reagents tested, followed by iodine. p-Chloromercuribenzoate showed a weak inhibitory effect. Diisopropylfluorophosphate and sodium chloride did not show any inhibitory effect on the enzyme. The beta-lactamase catalyzed the hydrolysis of methicillin and cloxacillin at two-thirds to one-third the rate of benzylpenicillin. On the other hand, the enzyme hydrolyzed cephalosporins and 7-methoxycephalosporin only slowly. With benzylpenicillin as a substrate, the Km increased sharply with decreasing pH and the pK alpha estimated from the Km versus pH curve was 6.5 to 7.0. In contrast, with cloxacillin as a substrate, the Km showed a minimum at pH 7.5. The Vmax changed with pH in a bell-shaped curve in the case of benzylpenicillin, but the Vmax for cloxacillin changed only within a small range. In addition, the ratio of the hydrolysis rate of benzylpenicillin and cloxacillin at 30 degrees C and 20 degrees C (V30 degrees/V20 degrees) was found to be 1.23 and 1.55, respectively. These results indicate that the S. cacaoi beta-lactamase behaves differently toward benzylpenicillin and cloxacillin, although both are penicillins. S. cacaoi seems to release beta-lactamase into the culture medium soon after its biosynthesis without retaining it in the membrane and the soluble fraction. The possible relationships between beta-lactamases from Streptomyces and those from pathogenic bacteria are discussed.     

Structural and kinetic studies on beta-lactamase K1 from Klebsiella aerogenes.

beta-Lactamase K1 from Klebsiella aerogenes 1082E hydrolyses both penicillins and cephalosporins comparably and is inhibited by mercurials but not by cloxacillin. These properties distinguish it from those other beta-lactamases that have been allotted to classes on the basis of their amino sequences. beta-Lactamase K1 has been isolated by affinity chromatography; its composition shows resemblances to class A beta-lactamases. Moreover, the N-terminal sequence is similar to those of class A beta-lactamases: there is about 30% identity over the first 32 residues. Furthermore, a putative active-site octapeptide has been isolated and its sequence is similar to the region around the active-site serine residue in class A beta-lactamases. There is one thiol group in beta-lactamase K1; it is not essential for activity. The pH-dependence of kcat. and kcat./Km for the hydrolysis of benzylpenicillin by beta-lactamase K1 were closely similar, suggesting that the rate-determining step is cleavage of the beta-lactam ring.     

'Beta-lactams' as beta-lactamase inhibitors

The application of inhibitors to block the beta-lactamase destruction of penicillins and cephalosporins by resistant bacteria is a potentially useful way of improving the efficacy of established compounds. Certain semi-synthetic penicillins and cephalosporins have been found to be competitive inhibitors of selected beta-lactamases but an examination of streptomycete culture fluids has revealed two new types of beta-lactam compound: clavulanic acid, which is a progressive inactivator of a wide range of beta-lactamases, and the olivanic acids, which are both broad-spectrum antibiotics and potent beta-lactamase inhibitors. Penicillanic acid sulphone and 6-beta-bromopenicillanic acid have been shown to be significant inhibitors of beta-lactamase. The chemotherapeutic application of these compounds is discussed.     

Site-saturation mutagenesis and three-dimensional modelling of ROB-1 define a substrate binding role of Ser130 in class A beta-lactamases.

Site-saturation mutagenesis was performed on the class A ROB-1 beta-lactamase at conserved Ser130, which is centrally located in the antibiotic binding site where it can participate in both protein-protein and protein-substrate hydrogen bonding. Mutation Thr130 gave a beta-lactamase hydrolysing penicillins and cephalosporins but which showed a 3-fold lower affinity (Km) for ampicillin and cephalexin, and a 30-fold lower hydrolytic (Vmax) activity for ampicillin. In contrast, the hydrolytic activity for cephalexin was similar to the wild-type for the Thr130 mutation. Mutation Gly130 gave a beta-lactamase hydrolysing only penicillins with an affinity and hydrolysis activity for these compounds approximately 15-fold lower than the wild-type, but no detectable activity against cephalosporins. Mutation Ala130 produced an enzyme capable of hydrolysing penicillins only at a low rate. Modelling the ROB-1 active site was done from the refined 2 A X-ray structure of the homologous Bacillus licheniformis beta-lactamase. Ampicillin and cephalexin were docked into the active site and were energy minimized with the CVFF empirical force field. Dockings were stable only when Ser70 was made anionic and Glu166 was made neutral. Interaction energies and distances were calculated for fully hydrated pre-acylation complexes with the Ser, Thr, Gly and Ala130 enzymes. The catalytic data from all mutations and the computed interactions from modelling confirmed that the Ser130 has a structural as well as a functional role in binding and hydrolysis of penicillins. This highly conserved residue also plays a substrate specificity role by hydrogen binding the carboxylic acid group of cephalosporins more tightly than penicillins.     

Purification and characterization of a beta-lactamase from Haemophilus ducreyi in Escherichia coli

A pCb plasmid encoding a beta-lactamase from Haemophilus ducreyi was transferred to Escherichia coli, purified, and characterized. The beta-lactamase could be isolated from a culture filtrate and further purified by ammonium sulfate and chelating Sepharose fast flow loaded with Zn(2+). The purified enzyme resulted in a major band at approximately 30-kDa on SDS-PAGE and its pI was determined to be 5.4. The beta-lactamase could hydrolyze both penicillin antibiotics including ampicillin, benzylpenicillin, and carbenicillin as well as cephalosporin antibiotics including nitrocefin, cephalothin, cephaloridine, and cefoperazone. However, benzylpenicillin was the best substrate. The enzyme activity was inhibited by clavulanic acid but not by boric acid, cefotaxime, ethylenediaminetetraacetic acid, or phenylmethylsulfonyl fluoride. The sequence of the beta-lactamase gene was also determined. It confirmed that the enzyme belonged to a class A beta-lactamase which had 99% identity to the ampicillin resistance transposon Tn3 of pBR322. Two nucleotides were different between the E. coli (Tn3) and H. ducreyi (pCb) genes that affected the amino-acid sequence. The valine at position 82 (ABL 84) was changed to isoleucine and the alanine at position 182 (ABL 184) was changed to valine. Genetic homogeneity among beta-lactamases is remarkable. Amino acid sequencing of some beta-lactamases has shown that substitution of only a few amino acids in the bla gene leads to high-level resistance against specific cephalosporins.     

Hydrolysis of third-generation cephalosporins by class C beta-lactamases. Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes

Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics. An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria. The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase. Unlike wild-type (WT) forms, such as the E. cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime. To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-[(Z)-methoxyimino]acetylaminomethyl phosphonate has been synthesized and characterized. This phosphonate was designed to generate a transition state analog for turnover of cefotaxime. The crystal structures of complexes of the phosphonate with both ES GC1 and WT C. freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms). The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme. In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed. In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried. During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule. The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.     

Engineering a novel beta-lactamase by a single point mutation

beta-Lactamases are widespread and efficient bacterial enzymes which play a major role in bacterial resistance to penicillins and cephalosporins. In order to elucidate the role of the residues lying in a conserved loop of the enzymatic cavity of the active-site serine Streptomyces albus G beta-lactamase, modified proteins were produced by oligo-directed mutagenesis. Mutation of Asn116, which lies on one side of the active site cavity pointing to the substrate-binding site, into a serine residue resulted in spectacular modifications of the specificity profile of the enzyme. That replacement yielded an enzyme with a nearly unchanged activity towards good penicillin substrates. In sharp contrast its efficiency in hydrolysing cephalosporins was drastically reduced, the best substrates suffering the largest decrease in the second-order rate constant for serine acylation. In fact that single mutation generated a truly new enzyme behaving exclusively as a penicillinase, a situation which is never encountered to the same degree in any of the numerous naturally occurring variants of class A beta-lactamases.