Kinetics and mechanism of inactivation of the RTEM-2 beta-lactamase by phenylpropynal. Identification of the characteristic chromophore

beta-Lactamases of all three classes, A, B, and C, are inactivated by phenylpropynal and p-nitrophenylpropynal. The inactivation of RTEM-2 beta-lactamase and of Bacillus cereus beta-lactamase I is accelerated in the presence of A type substrates such as dicloxacillin, quinacillin, and cefoxitin, which are thought to expand or loosen the conformation of these enzymes. In the presence and absence of cefoxitin the inactivation of the RTEM-2 beta-lactamase is first and second order, respectively, in phenylpropynal concentration. The additional phenylpropynal molecule in the latter case may serve the same function as cefoxitin, viz. catalyze access to sensitive functional groups. Correlation of the loss of activity of the RTEM-2 enzyme with the extent of modification suggests that the modification of any one of about four kinetically equivalent groups leads to inactivation. Modification of all of the above mentioned enzymes leads to formation of a characteristic chromophore of unusual stability to nucleophiles, which absorbs maximally between 315 and 320 nm. A consideration of the properties of model compounds demonstrated that the protein-bound chromophore is that of a 1-phenyl-3-imino-1-propen-1-ammonium ion (Formula: see text), formed by reaction of phenylpropynal with two enzymic amine groups, and thus cross-linking the enzyme intramolecularly. Phenylpropynal may be a convenient general reagent for rapid and stable intramolecular cross-linking of proteins through lysine.     

Active sites of beta-lactamases from Bacillus cereus

There are two extracellular beta-lactamases produced by Bacillus cereus 569. One of these enzymes, beta-lactamase I, is inactivated by 6-beta-bromopenicillanic acid: the site of reaction is serine-44. This is a conserved amino acid residue in the other beta-lactamases whose structures have been determined, and it becomes a good candidate for an active-site group in these enzymes. The inactivation may involve a rearrangement leading to a dihydrothiazine. The other extracellular enzyme produced by B. cereus, beta-lactamase II, is exceptional in requiring metal ions for activity. The Zn II and Co II enzymes (the former is more active) have been studied by nuclear magnetic resonance, and by absorption spectroscopy. The groups that bind the metal ion required for activity are three histidine residues and the enzyme's sole thiol group.     

Cloning and sequencing of the metallothioprotein beta-lactamase II gene of Bacillus cereus 569/H in Escherichia coli

The structural gene for beta-lactamase II (EC 3.5.2.6), a metallothioenzyme, from Bacillus cereus 569/H (constitutive for high production of the enzyme) was cloned in Escherichia coli, and the nucleotide sequence was determined. This is the first class B beta-lactamase whose primary structure has been reported. The amino acid sequence of the exoenzyme form, deduced from the DNA, indicates that beta-lactamase II, like other secreted proteins, is synthesized as a precursor with a 30-amino acid N-terminal signal peptide. The pre-beta-lactamase II (Mr, 28,060) is processed in E. coli and in B. cereus to a single mature protein (Mr, 24,932) which is totally secreted by B. cereus but in E. coli remains intracellular, probably in the periplasm. The expression of the gene in E. coli RR1 on the multicopy plasmid pRWHO12 was comparable to that in B. cereus, where it is presumably present as a single copy. The three histidine residues that are involved (along with the sole cysteine of the mature protein) in Zn(II) binding and hence in enzymatic activity against beta-lactams were identified. These findings will help to define the secondary structure, mechanism of action, and evolutionary lineage of B. cereus beta-lactamase II and other class B beta-lactamases.     

Interaction of beta-iodopenicillanate with the beta-lactamases of Streptomyces albus G and Actinomadura R39.

The beta-lactamases of Streptomyces albus G and Actinomadura R39 are inactivated by beta-iodopenicillanate. However, in contrast with the beta-lactamase I from Bacillus cereus, they also efficiently catalyse the hydrolysis of the inactivator; with the S. albus G enzyme, kcat. is larger than 25s-1 and the number of turnovers before inactivation is 515. With the A. R39 enzyme, kcat. is larger than 50s-1 and the number of turnovers before inactivation is 80. After hydrolysis of the beta-lactam amide bond, the product rearranges into 2.3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which exhibits an absorption maximum at 305 nm.     

Clavulanic acid inactivation of SHV-1 and the inhibitor-resistant S130G SHV-1 beta-lactamase. Insights into the mechanism of inhibition

Clavulanic acid is a potent mechanism-based inhibitor of TEM-1 and SHV-1beta-lactamases, enzymes that confer resistance to beta-lactams in many gram-negative pathogens. This compound has enjoyed widespread clinical use as part of beta-lactam beta-lactamase inhibitor therapy directed against penicillin-resistant pathogens. Unfortunately, the emergence of clavulanic acid-resistant variants of TEM-1 and SHV-1 beta-lactamase significantly compromise the efficacy of this combination. A single amino acid change at Ambler position Ser130 (Ser --> Gly) results in resistance to inactivation by clavulanate in the SHV-1 and TEM-1beta-lactamases. Herein, we investigated the inactivation of SHV-1 and the inhibitor-resistant S130G variant beta-lactamases by clavulanate. Using liquid chromatography electrospray ionization mass spectrometry, we detected multiple modified proteins when SHV-1 beta-lactamase is inactivated by clavulanate. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to study tryptic digests of SHV-1 and S130Gbeta-lactamases (+/- inactivation with clavulanate) and identified peptides modified at the active site Ser70. Ultraviolet (UV) difference spectral studies comparing SHV-1 and S130Gbeta-lactamases inactivated by clavulanate showed that the formation of reaction intermediates with absorption maxima at 227 and 280 nm are diminished and delayed when S130Gbeta-lactamase is inactivated. We conclude that the clavulanic acid inhibition of the S130G beta-lactamase must follow a branch of the normal inactivation pathway. These findings highlight the importance of understanding the intermediates formed in the inactivation process of inhibitor-resistant beta-lactamases and suggest how strategic chemical design can lead to novel ways to inhibit beta-lactamases.     

Beta-lactamase III of Bacillus cereus 569: membrane lipoprotein and secreted protein

A third beta-lactamase in Bacillus cereus 569 has been identified and characterized. It corresponds to gamma-penicillinase reported by Pollock [Pollock, M. R. (1956) J. Gen. Microbiol. 15, 154-169] but whose existence has been questioned since then. It will be called beta-lactamase III. It resembles the class A beta-lactamases but is immunologically distinct from the major class A secreted beta-lactamase I of B. cereus. As with several other Gram-positive beta-lactamases it occurs in two forms, membrane bound as a glyceride-cysteine lipoprotein and as a hydrophilic secreted protein formed by cleavage on the carboxyl side of the modified cysteine that is the membrane attachment site. It is produced in all B. cereus 569 strains tested but is absent in B. cereus 5/b. Antibody to beta-lactamase III interacts to varying degrees with all the known class A beta-lactamases, most strongly with that of B. licheniformis 749/C.     

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.     

Hyperexpression in Escherichia coli, purification, and characterization of the metallo-beta-lactamase of Bacillus cereus 5/B/6.

We used site-directed mutagenesis to introduce both a NdeI restriction endonuclease site and an initiator codon at the junction of the leader and structural gene sequences of the metallo-beta-lactamase of Bacillus cereus 5/B/6. This construct allowed us to clone just the beta-lactamase structural gene sequence into an Escherichia coli expression vector. E. coli cells were transformed with the recombinant plasmid, the B. cereus beta-lactamase was expressed, and these E. coli cells were disrupted by sonic oscillation. When the resultant suspensions were clarified by ultracentrifugation, the B. cereus beta-lactamase represented 15% of the total protein in the supernatant. Subsequent gel filtration and ion-exchange chromatography allowed the first reported purification to homogeneity of the B. cereus beta-lactamase from E. coli with an 87% recovery and an overall yield of 17 mg of enzyme per liter of cell culture. The electrophoretic mobilities of the enzyme expressed in and purified from E. coli and the enzyme purified directly from B. cereus were identical in both native and sodium dodecyl sulfate gel electrophoreses. As with the B. cereus enzyme, Km and Vmax (using cephalosporin C as substrate) for the enzyme purified from E. coli were 0.39 mM and 1333 units/mg protein, respectively. Likewise, the Co(II)-reconstituted enzyme purified from E. coli, which retained 29% of the activity of the Zn(II) enzyme, had electronic absorption spectra with maxima at 347, 551, 617, and 646 nm with extinction coefficients of 900, 250, 173, and 150 M-1 cm-1, respectively.     

Processing of Bacillus cereus 569/H beta-lactamase I in Escherichia coli and Bacillus subtilis

The gene for Bacillus cereus 569/H beta-lactamase I, penPC, has recently been cloned and sequenced (Mézes, P. S. F., Yang, Y. Q., Hussain, M., and Lampen, J. O. (1983) FEBS Lett. 161, 195-200). A typical prokaryotic signal peptide but with no lipoprotein modification site, as present in the Bacillus licheniformis 749/C beta-lactamase, was indicated by the DNA sequence for this secretory protein. We have here purified the beta-lactamase I products found in Escherichia coli and Bacillus subtilis carrying penPC and have determined the first 20 NH2-terminal amino acids of each of the forms. Processing of the beta-lactamase I in E. coli occurs at a single site which is characteristic for cleavage by a signal peptidase. B. subtilis secreted two distinct products to the culture medium which were both smaller than the single product formed in E. coli. Sequencing of [35S]Met-labeled pre-beta-lactamase I from phenylethyl alcohol-treated cells of B. cereus 569/H indicated that UUG is being utilized as the initiation codon for penPC. The same result was obtained for the pre-beta-lactamase I from similarly treated cells of the closely related B. cereus 5/B strain.     

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii,Citrobacter murliniae,Citrobacter werkmanii,Escherichia fergusonii and Enterobacter cancerogenus

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.     

Sensitivity of 5 beta-lactam antibiotics to beta-lactamase isolated from E. coli and its respective transconjugates

54 beta-lactamase producing E. coli were tested to observe their eventual capacity to transfer beta-lactamase production by conjugation to a receiving E. coli K12 C600 Na-. About 16% (9/54) of these strains transferred beta-lactamase producing capacity. MICs of five beta-lactam antibiotics (Ampicillin, Cephaloridine, Cephalexine, Cefuroxime, Cefotaxime) were performed against E. coli donors and E. coli K12 C600 transconjugates. It was observed a remarkable increase only of Ampicillin MICs against all transconjugates++. Beta-lactamases produced by donors and transconjugants were isolated and purified by sonication and high speed centrifugation. Sensitivity of the six antibiotics to these purified beta-lactamases was assessed by a spectrophotometric method that utilizes the velocity of cytochrome c reduction. beta-lactamases produced by transconjugants have identical substrate profile that beta-lactamases produced by donors.     

Characterization of the gene encoding the beta-lactamase of the psychrophilic marine bacterium Moritella marina strain MP-1

The beta-lactamase gene (mbla) of the psychrophilic marine bacterium Moritella marina strain MP-1 was identified in a previously isolated genomic DNA fragment and it was expressed in Escherichia coli cells. The mbla gene encoded a protein consisting of 287 amino acid residues. Its predicted amino acid sequence showed approximately 50% identity with that of a number of class A beta-lactamases, especially with that of CARB/PSE type of beta-lactamases (carbenicillinases). E. coli transformed with the plasmid containing mbla grew on an ampicillin-containing plate at 37 degrees C but not at 42 degrees C, suggesting that the beta-lactamase of this bacterium is heat-labile.     

Tazobactam inactivation of SHV-1 and the inhibitor-resistant Ser130 -->Gly SHV-1 beta-lactamase: insights into the mechanism of inhibition

The increasing number of bacteria resistant to combinations of beta-lactam and beta-lactamase inhibitors is creating great difficulties in the treatment of serious hospital-acquired infections. Understanding the mechanisms and structural basis for the inactivation of these inhibitor-resistant beta-lactamases provides a rationale for the design of novel compounds. In the present work, SHV-1 and the Ser(130) --> Gly inhibitor-resistant variant of SHV-1 beta-lactamase were inactivated with tazobactam, a potent class A beta-lactamase inhibitor. Apoenzymes and inhibited beta-lactamases were analyzed by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), digested with trypsin, and the products resolved using LC-ESI/MS and matrix-assisted laser desorption ionization-time of flight mass spectrometry. The mass increases observed for SHV-1 and Ser(130) --> Gly (+ Delta 88 Da and + Delta 70 Da, respectively) suggest that fragmentation of tazobactam readily occurs in the inhibitor-resistant variant to yield an inactive beta-lactamase. These two mass increments are consistent with the formation of an aldehyde (+ Delta 70 Da) and a hydrated aldehyde (+ Delta 88 Da) as stable products of inhibition. Our results reveal that the Ser --> Gly substitution at amino acid position 130 is not essential for enzyme inactivation. By examining the inhibitor-resistant Ser(130) --> Gly beta-lactamase, our data are the first to show that tazobactam undergoes fragmentation while still attached to the active site Ser(70) in this enzyme. After acylation of tazobactam by Ser(130) --> Gly, inactivation proceeds independent of any additional covalent interactions.     

Production, purification and spectral properties of metal-dependent beta-lactamases of Bacillus cereus

New methods for the production of consistently high levels of metal-dependent beta-lactamases (beta-lactamhydrolase, EC 3.5.2.6) from strains 569/H/9 and 5/B/6 of Bacillus cereus are described which have significant advantages over those reported previously. For example, these techniques do not require a fermentor with pH-stat capabilities. We also describe rapid very-high-yield purification schemes for the metal-dependent beta-lactamases from these strains, employing high-performance ultrafiltration (HPUF) and mass ion exchange techniques. Furthermore, we have developed improved methods for the removal of the active site Zn(II) and reconstitution of the beta-lactamase enzymatic activity with Co(II), which result in higher recovery of the original activity than previously reported. In order to characterize the purified beta-lactamases II of B. cereus 569/H/9 and 5/B/6 we have examined the molecular weights, and steady state kinetic parameters of Zn(II) enzymes, and the electronic and EPR spectra of the Co(II)-reconstituted enzymes. EPR spectra of CO(II)-reconstituted beta-lactamase from B. cereus 5/B/6 have not been previously reported.     

Probing active site chemistry in SHV beta-lactamase variants at Ambler position 244. Understanding unique properties of inhibitor resistance

Inhibitor-resistant class A beta-lactamases are an emerging threat to the use of beta-lactam/beta-lactamase inhibitor combinations (e.g. amoxicillin/clavulanate) in the treatment of serious bacterial infections. In the TEM family of Class A beta-lactamases, single amino acid substitutions at Arg-244 confer resistance to clavulanate inactivation. To understand the amino acid sequence requirements in class A beta-lactamases that confer resistance to clavulanate, we performed site-saturation mutagenesis of Arg-244 in SHV-1, a related class A beta-lactamase found in Klebsiella pneumoniae. Twelve SHV enzymes with amino acid substitutions at Arg-244 resulted in significant increases in minimal inhibitory concentrations to ampicillin/clavulanate when expressed in Escherichia coli. Kinetic analyses of SHV-1, R244S, R244Q, R244L, and R244E beta-lactamases revealed that the main determinant of clavulanate resistance was reduced inhibitor affinity. In contrast to studies in the highly similar TEM enzyme, we observed increases in clavulanate k(inact) for all mutants. Electrospray ionization mass spectrometry of clavulanate inhibited SHV-1 and R244S showed nearly identical mass adducts, arguing against a difference in the inactivation mechanism. Testing a wide range of substrates with C3-4 carboxylates in different stereochemical orientations, we observed impaired affinity for all substrates among inhibitor resistant variants. Lastly, we synthesized two boronic acid transition state analogs that mimic cephalothin and found substitutions at Arg-244 markedly affect both the affinity and kinetics of binding to the chiral, deacylation transition state inhibitor. These data define a role for Arg-244 in substrate and inhibitor binding in the SHV beta-lactamase.     

Understanding resistance to beta-lactams and beta-lactamase inhibitors in the SHV beta-lactamase: lessons from the mutagenesis of SER-130

Bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations by single amino acid mutations in class A beta-lactamases threatens our most potent clinical antibiotics. In TEM-1 and SHV-1, the common class A beta-lactamases, alterations at Ser-130 confer resistance to inactivation by the beta-lactamase inhibitors, clavulanic acid, and tazobactam. By using site-saturation mutagenesis, we sought to determine the amino acid substitutions at Ser-130 in SHV-1 beta-lactamase that result in resistance to these inhibitors. Antibiotic susceptibility testing revealed that ampicillin and ampicillin/clavulanic acid resistance was observed only for the S130G beta-lactamase expressed in Escherichia coli. Kinetic analysis of the S130G beta-lactamase demonstrated a significant elevation in apparent Km and a reduction in kcat/Km for ampicillin. Marked increases in the dissociation constant for the preacylation complex, KI, of clavulanic acid (SHV-1, 0.14 microm; S130G, 46.5 microm) and tazobactam (SHV-1, 0.07 microm; S130G, 4.2 microm) were observed. In contrast, the k(inact)s of S130G and SHV-1 differed by only 17% for clavulanic acid and 40% for tazobactam. Progressive inactivation studies showed that the inhibitor to enzyme ratios required to inactivate SHV-1 and S130G were similar. Our observations demonstrate that enzymatic activity is preserved despite amino acid substitutions that significantly alter the apparent affinity of the active site for beta-lactams and beta-lactamase inhibitors. These results underscore the mechanistic versatility of class A beta-lactamases and have implications for the design of novel beta-lactamase inhibitors.     

Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A beta-lactamase Toho-1

The production of class A beta-lactamases is a major cause of clinical resistance to beta-lactam antibiotics. Some of class A beta-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum beta-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum beta-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.     

The pH-dependence of class B and class C beta-lactamases.

The classification by structure allots beta-lactamases to (at present) three classes, A, B and C. The pH-dependence of the kinetic parameters for class B and class C have been determined. They differ from each other and from class A beta-lactamases. The class B enzyme was beta-lactamase II from Bacillus cereus 569/H/9. The plots of kcat against pH for the hydrolysis of benzylpenicillin by Zn(II)-requiring beta-lactamase II and Co(II)-requiring beta-lactamase II were not symmetrical, but those of kcat/Km were. A similar feature was observed for the hydrolysis of both benzylpenicillin and cephalosporin C by a class C beta-lactamase from Pseudomonas aeruginosa. The results have been interpreted by a scheme in which two ionic forms of an intermediate can give product, but do so at differing rates.