Beta-lactamase Substrate Research Articles

Fecal Carriage of Extended-Spectrum beta-Lactamase Producing Escherichia coli among Health Science Students.

The incidence of antibiotic resistance in commensal bacteria is increasing with the production of extended-spectrum beta-lactamase. Therefore, this study was conducted to understand the status of fecal carriage of such enzyme producing Escherichia coli among health science students of seven different faculties of Institute of Medicine, Tribhuvan University. This was a cross-sectional study conducted over six months among the health science students. One stool sample collected from each student was cultured and Escherichia coli isolates were identified, antibiotic sensitivity profile was produced, and extended-spectrum beta-lactamase production was detected following Clinical and Laboratory Standards Institute guidelines. A total of 156 students participated in the study, and Escherichia coli was isolated from all. Out of the total 156 Escherichia coli isolates, 11.5% were extended-spectrum beta-lactamase-producers and 14.7% were multidrug-resistant. The highest rate of fecal carriage of extended-spectrum beta-lactamase-producing Escherichia coli was found among Bachelor of Medicine and Bachelor of Surgery students (17.5%) and Bachelor of Science in Medical Imaging Technology (16.7%) students. Such enzyme producing Escherichia coli was found in the range of 6.9% to 25.0% among second- to fifth-year students. A significant number of extended-spectrum beta-lactamase-producing isolates were resistant to ciprofloxacin and gentamicin, apart from other extended-spectrum beta-lactamase substrate antibiotics, when compared with non-producers. A high rate of extended-spectrum beta-lactamase-producing Escherichia coli was detected from the gut of healthy health science students which indicates their possible dissemination throughout the wider community resulting in potential outbreak of infections caused by such organisms.

Recognition and interaction of CphA from Aeromonas hydrophila with imipenem and biapenem by spectroscopic analysis in combination with molecular docking.

The molecular recognition and interaction of CphA from Aeromonas hydrophila with imipenem (Imip) and biapenem (Biap) were studied by means of the combined use of fluorescence spectra and molecular docking. The results showed that both the fluorescence quenching of CphA by Imip and Biap were caused through the combined dynamic and static quenching, and the latter was dominating in the process; the microenvironment and conformational of CphA were altered upon the addition of Imip and Biap from synchronous and three-dimensional fluorescence. The binding of CphA with Imip or Biap caused a conformational change in the loop of CphA, and through the conformational change, the loop opened the binding pocket of CphA to allow for an induced fit of the newly introduced ligand. In the binding of CphA with Imip, the whole molecule entered into the active pocket of CphA. The binding was driven by enthalpy change, and the binding force between them was mainly hydrogen bonding and Van der Waals force; whereas in the binding of CphA with Biap, only the beta-lactam ring of Biap entered into the binding pocket of CphA while the side chain was located outside the active pocket. The binding was driven by the enthalpy change and entropy change together, and the binding force between them was mainly electrostatic interaction. This study provided an insight into the recognition and binding of CphA with antibiotics, which may be helpful for designing new substrate for beta-lactamase and developing new antibiotics resistant to superbugs.

Novel Chromogenic Substrate for Bacterial β-Lactamases Based on Cephalosporin Modified with an Epoxy Group

Beta-lactamases are the key enzymes involved in resistance to beta-lactam antibiotics in pathogenic bacteria causing infectious diseases. The search for new inhibitors and the study of the resistance mechanisms require the production of chromogenic substrates for beta-lactamases. A novel cephalosporin derivative with an epoxy functional group named CMPD1 is synthesized. It is shown to be a substrate for TEM type beta-lactamases, which is hydrolyzed to form a colored product. The hydrolysis product has an optical absorption maximum at 450 nm. The difference in the absorption maxima of the substrate and the product is 95 nm, and, therefore, CMPD1 exceeds the previously described substrates, according to this parameter. It has been found that the CMPD1 compound is hydrolyzed only by the TEM type beta-lactamases that lack mutations in the active site. This can be used to study the mechanisms of the catalytic effect of beta-lactamases.

Effect of synthetic and natural phospholipids on N-acylphosphatidylethanolamine-hydrolyzing phospholipase D activity

N-Acylethanolamines (NAEs) constitute a family of endogenous bioactive lipids that includes arachidonoylethanolamide (anandamide), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). These lipids are formed from their respective N-acylated ethanolamine phospholipid (NAPE) precursor by the action of a phospholipase D enzyme (NAPE-PLD). Anandamide, OEA, and PEA are all bioactive lipids that may influence, amongst others: neuroinflammation, food intake, and oocyte implantation. Here we have synthesized a number of NAPE analogues with variation in the phosphoester structure. The NAPE analogues as well as selected phospholipids and beta-lactamase substrates were tested as potential modifiers of cloned human NAPE-PLD in an enzyme assay involving a 14C-labeled diether-NAPE substrate. One hit was identified, namely 1,2-dihexanoyl-glycero- N-(3-(tetradecanoylamino)propyl)phosphoramidate (AHP-71B) which showed inhibitory activity and may serve as template for further structure–activity developments. Furthermore, it was found that NAPE-PLD was activated by phosphatidylethanolamine and inhibited by the beta-lactamase substrate nitrocefin.

HTS-Compatible β-Lactamase Transcriptional Reporter Gene Assay for Interrogating the Heat Shock Response Pathway

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR.

β-Lactamase Reporter System for Selecting High-Producing Yeast Clones

In modern production of protein biopharmaceuticals, a good screening and selection method of high-producing clones can dramatically influence the whole production process and lead to lower production costs. We have created a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoris that is based on the beta-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use beta-lactamase activity as a measure of the expression level of the protein of interest. A novel expression vector with two independent expression cassettes was designed and tested using green fluorescent protein (GFP) as a model. The first cassette contained the GFP gene under the control of a strong, inducible AOX1 promoter, while the second cassette consisted of the beta-lactamase reporter gene under the control of a weak constitutive YPT1 promotor. High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the beta-lactamase substrate added to the medium. beta-lactamase activity was found to positively correlate with GFP fluorescence. The reporter system described is widely applicable-it can be easily applied to other, also pharmaceutically relevant proteins and to other yeast expression systems, such as Saccharomyces cerevisiae and Hansenula polymorpha.

Ceftobiprole

Ceftobiprole, an investigational cephalosporin, is currently in phase III clinical development. Ceftobiprole is a broad-spectrum cephalosporin with demonstrated in vitro activity against Gram-positive cocci, including meticillin-resistant Staphylococcus aureus (MRSA) and meticillin-resistant S. epidermidis, penicillin-resistant S. pneumoniae, Enterococcus faecalis, Gram-negative bacilli including AmpC-producing Escherichia coli and Pseudomonas aeruginosa, but excluding extended-spectrum beta-lactamase-producing strains. Like cefotaxime, ceftriaxone, ceftazidime, and cefepime, ceftobiprole demonstrates limited activity against anaerobes such as Bacteroides fragilis and non-fragilis Bacteroides spp. In single-step and serial passage in vitro resistance development studies, ceftobiprole demonstrated a low propensity to select for resistant subpopulations. Ceftobiprole, like cefepime, is a weak inducer and a poor substrate for AmpC beta-lactamases.Ceftobiprole medocaril, the prodrug of ceftobiprole, is converted by plasma esterases to ceftobiprole in <30 minutes. Peak serum concentrations of ceftobiprole observed at the end of a single 30-minute infusion were 35.5 mug/mL for a 500-mg dose and 59.6 mug/mL for a 750-mg dose. The volume of distribution of ceftobiprole is 0.26 L/kg ( approximately 18 L), protein binding is 16%, and its serum half-life is approximately 3.5 hours. Ceftobiprole is renally excreted ( approximately 70% in the active form) and systemic clearance correlates with creatinine clearance, meaning that dosage adjustment is required in patients with renal dysfunction. Ceftobiprole has a modest post-antibiotic effect (PAE) of approximately 0.5 hours for MRSA and a longer PAE of approximately 2 hours for penicillin-resistant pneumococci. Ceftobiprole, when administered intravenously at 500 mg once every 8 hours (2-hour infusion), has a >90% probability of achieving f T(>MIC) (free drug concentration exceeds the minimum inhibitory concentration [MIC]) for 40% and 60%, respectively, of the dosing interval for isolates with ceftobiprole MIC < or =4 and < or =2 mg/L, respectively.Currently, only limited clinical trial data are published for ceftobiprole. In a phase III trial, 784 patients with Gram-positive skin infections were randomized to treatment with either ceftobiprole 500 mg or vancomycin 1 g, each administered twice daily for 7-14 days; 93.3% of patients were clinically cured with ceftobiprole compared with 93.5% receiving vancomycin, and the eradication rate for MRSA infections was 91.8% for ceftobiprole compared with 90% for vancomycin. A phase III, randomized, double-blind, multicenter trial compared ceftobiprole 500 mg every 8 hours with vancomycin 1 g every 12 hours plus ceftazidime 1 g every 8 hours in patients with complicated skin and skin structure infections. Of the 828 patients enrolled, 31% had diabetic foot infections, 30% had abscesses, and 22% had wounds. No difference in clinical cure was reported in the clinically evaluable, intent-to-treat and microbiologically evaluable populations with cure rates of 90.5%, 81.9%, and 90.8%, respectively, in the ceftobiprole-treated patients and 90.2%, 80.8%, and 90.5%, respectively, in the vancomycin plus ceftazidime-treated group. Microbiologic eradication of Gram-positive cocci meticillin-susceptible S. aureus (MSSA) [ceftobiprole 91% vs vancomycin plus ceftazidime 92%] and MRSA (ceftobiprole 87% vs vancomycin plus ceftazidime 80%), as well as Gram-negative bacilli, E. coli (ceftobiprole 89% vs vancomycin plus ceftazidime 92%), and P. aeruginosa (ceftobiprole 87% vs vancomycin plus ceftazidime 100%), was not significantly different between groups. Similar cures rates in the microbiologically evaluable population occurred in both groups for Panton-Valentine leukocidin (PVL)-positive MSSA and PVL-positive MRSA.Currently, ceftobiprole has completed phase III trials for complicated skin and skin structure infections due to MRSA and nosocomial pneumonia due to suspected or proven MRSA; phase III trials are also ongoing in community-acquired pneumonia. Ceftobiprole has so far demonstrated a good safety profile in preliminary studies with similar tolerability to comparators. The broad-spectrum activity of ceftobiprole may allow it to be used as monotherapy in situations where a combination of antibacterials might be required. Further clinical studies are needed to determine the efficacy and safety of ceftobiprole and to define its role in patient care.

Different Transition-State Structures for the Reactions of β-Lactams and Analogous β-Sultams with Serine β-Lactamases

Beta-sultams are the sulfonyl analogues of beta-lactams, and N-acyl beta-sultams are novel inactivators of the class C beta-lactamase of Enterobacter cloacae P99. They sulfonylate the active site serine residue to form a sulfonate ester which subsequently undergoes C-O bond fission and formation of a dehydroalanine residue by elimination of the sulfonate anion as shown by electrospray ionization mass spectroscopy. The analogous N-acyl beta-lactams are substrates for beta-lactamase and undergo enzyme-catalyzed hydrolysis presumably by the normal acylation-deacylation process. The rates of acylation of the enzyme by the beta-lactams, measured by the second-order rate constant for hydrolysis, kcat/K(m), and those of sulfonylation by the beta-sultams, measured by the second-order rate constant for inactivation, k(i), both show a similar pH dependence to that exhibited by the beta-lactamase-catalyzed hydrolysis of beta-lactam antibiotics. Electron-withdrawing groups in the aryl residue of the leaving group of N-aroyl beta-lactams increase the rate of alkaline hydrolysis and give a Bronsted beta(lg) of -0.55, indicative of a late transition state for rate-limiting formation of the tetrahedral intermediate. Interestingly, the corresponding Bronsted beta(lg) for the beta-lactamase-catalyzed hydrolysis of the same substrates is -0.06, indicative of an earlier transition state for the enzyme-catalyzed reaction. By contrast, although the Bronsted beta(lg) for the alkaline hydrolysis of N-aroyl beta-sultams is -0.73, similar to that for the beta-lactams, that for the sulfonylation of beta-lactamase by these compounds is -1.46, compatible with significant amide anion expulsion/S-N fission in the transition state. In this case, the enzyme reaction displays a later transition state compared with hydroxide-ion-catalyzed hydrolysis of the beta-sultam.

Fluorescence resonance energy transfer-based HIV-1 virion fusion assay.

The fluorescence resonance energy transfer (FRET)-based HIV-1 virion fusion assay exploits the incorporation of beta-lactamase-Vpr chimeric proteins into HIV-1 virions and their subsequent delivery into the cytoplasm of target cells as a marker of fusion. This transfer can be monitored by the enzymatic cleavage of the CCF2-AM dye, a fluorescent substrate of beta-lactamase (BlaM), loaded into the target cells. BlaM cleavage of the beta-lactam ring in CCF2-AM prevents the FRET between the coumarin and fluorescein moieties of the dye. This cleavage changes the fluorescence emission spectrum of CCF2-AM from green (520 nm) to blue (447 nm), and thus permits detection of fusion by fluorescence microscopy, flow cytometry, or UV photometry. This assay is simple and rapid to perform, and exhibits high sensitivity and specificity. Importantly, it can be applied to study HIV-1 virion fusion in primary cells and can be combined with immunostaining for subset discrimination in heterogeneous target cell populations. Finally, the assay can also be adapted to study fusion mediated by the envelope proteins from other viruses through the construction of HIV-1 pseudotypes.

The kinetic properties of the carboxy terminal domain of the Bacillus licheniformis 749/I BlaR penicillin-receptor shed a new light on the derepression of beta-lactamase synthesis.

To study the properties of the BlaR penicillin-receptor involved in the induction of the Bacillus licheniformisbeta-lactamase, the water-soluble carboxy terminal domain of the protein (BlaR-CTD) was overproduced in the periplasm of Escherichia coli JM105 and purified to protein homogeneity. Its interactions with various beta-lactam antibiotics were studied. The second-order acylation rate constants k2/K' ranged from 0.0017 to more than 1 micro M-1s-1 and the deacylation rate constants were lower than 4 x 10-5 s-1. These values imply a rapid to very rapid formation of a stable acylated adduct. BlaR-CTD is thus one of the most sensitive penicillin-binding proteins presently described. In the light of these results, the kinetics of beta-lactamase induction in Bacillus licheniformis were re-examined. When starting with a rather high cell density, a good beta-lactamase substrate such as benzylpenicillin is too sensitive to beta-lactamase-mediated hydrolysis to allow full induction. By contrast, a poor beta-lactamase substrate (7-aminocephalosporanic acid) can fully derepress beta-lactamase expression under conditions where interference of the antibiotic with cell growth is observed. These results suggest that acylation of the penicillin receptor is a necessary, but not sufficient, condition for full induction.

Identification and characterization of an antibacterial peptide of the 26-kDa protease of Sarcophaga peregrina with antibacterial activity.

Previously, we purified a serine protease with a molecular mass of 26 kDa that exhibits potent antibacterial activity from a pupal extract of Sarcophaga peregrina (flesh fly). We divided this protease into 12 peptides and examined their antibacterial activity. A peptide corresponding to residues 155 to 174 (peptide 9) was found to exhibit antibacterial activity comparable to that of the 26-kDa protease. When Escherichia coli was treated with peptide 9, the permeability of both the outer and inner membranes increased, and substrates for beta-lactamase and beta-galactosidase entered the cells, but beta-galactosidase did not leak out of the cells under these conditions. It was suggested that residues 6 to 18 of peptide 9 form an amphiphilic alpha-helix under hydrophobic conditions with an N-terminal basic loop and then interact with acidic phospholipids in the bacterial membranes.

Synthesis and Reactivity with beta-Lactamases of "Penicillin-like" Cyclic Depsipeptides.

Several 7-carboxy-3-amido-3,4-dihydro-2H-1-benzopyran-2-ones have been synthesized as potential beta-lactamase substrates and/or mechanism-based inhibitors. Substituted o-tyrosine precursors were prepared by the Sörensen method and then heated in vacuo to give the lactones. These compounds are cyclic analogues of aryl phenaceturates which are known to be beta-lactamase substrates. The goal of incorporating the scissile ester group into a lactone was to retain the leaving group tethered to the acyl moiety at the acyl-enzyme stage of turnover by serine beta-lactamases, in a manner similar to that during penicillin turnover. Further, in two cases, a functionalized methylene group para to the leaving group phenoxide oxygen was incorporated. These molecules possess a latent p-quinone methide electrophile which could, in principle, be unmasked during enzymic turnover and react with an active site nucleophile. All of these compounds were found to be substrates of class A and C beta-lactamases, the first delta-lactones with such activity. Generally, k(cat) values were smaller than for the analogous acyclic depsipeptides, which suggests that the tethered leaving group may obstruct the attack of water on the acyl-enzymes. Further exploration of this structural theme might lead to quite inert acyl-enzymes and thus to significant inhibitors. Despite the apparent advantage offered by the longer-lived acyl-enzymes, the functionalized compounds were no better as irreversible inhibitors than comparable acyclic compounds [Cabaret, D.; Liu, J.; Wakselman, M.; Pratt, R. F.; Xu, Y. Bioorg. Med. Chem. 1994, 2, 757-771]. Thus, even tethered quinone methides, at least when placed as dictated by the structures of the present compounds, were unable to efficiently trap a nucleophile at serine beta-lactamase active sites.

When drug inactivation renders the target irrelevant to antibiotic resistance: a case story with beta-lactams.

By challenging the efficiency of some of our most useful antimicrobial weapons, bacterial antibiotic resistance is becoming an increasingly worrying clinical problem. A good antibiotic is expected to exhibit a high affinity for its target and to reach it rapidly, while escaping chemical modification by inactivating enzymes and elimination by efflux mechanisms. A study of the behaviour of a beta-lactamase-overproducing mutant of Enterobacter cloacae in the presence of several penicillins and cephalosporins showed that the minimum inhibitory concentration (MIC) values for several compounds were practically independent of the sensitivity of the target penicillin binding protein (PBP), even for poor beta-lactamase substrates. This apparent paradox was explained by analysing the equation that relates the antibiotic concentration in the periplasm to that in the external medium. Indeed, under conditions that are encountered frequently in clinical isolates, the factor characterizing the PBP sensitivity became negligible. The conclusions can be extended to all antibiotics that are sensitive to enzymatic inactivation and efflux mechanisms and must overcome permeability barriers. It would be a grave mistake to neglect these considerations in the design of future antibacterial chemotherapeutic agents.

Functional mimicry: elicitation of a monoclonal anti-idiotypic antibody hydrolizing beta-lactams.

Antigen mimicry by anti-idiotypic antibodies is investigated as a reliable strategy to achieve molecular imprinting of an enzymatic activity. A monoclonal anti-idiotypic antibody (Ab2-9G4H9) was elicited by using a monoclonal antibody (Ab1-7AF9) specific for the beta-lactamase active site. Catalytic features of Ab2 were characterized with beta-lactamase substrates. The antibody combining site appeared to have retained a part of the catalytic specificity. The relevance of the idiotypic mimicry concept for the generation of catalytic antibodies was further demonstrated by eliciting a third generation antibody (Ab3), which was shown to recognize beta-lactamase: the complete internal image properties of Ab2 9G4H9, including binding and catalytic properties, were thus checked.

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

The clinical isolate Escherichia coli PEY was highly resistant to amoxycillin, ticarcillin and piperacillin associated to beta-lactamase inhibitors such as clavulanic acid, sulbactam, tazobactam and brobactam but susceptible to cephalosporins, aztreonam and imipenem. The susceptibility to mecillinam indicated that this phenotype was not related to hyperproduction of the TEM-1 beta-lactamase. E. coli PEY produced a new plasmid-mediated inhibitor-resistant beta-lactamase of pI 5.2, which was named IRT-4. The determination of the amino acid sequence (Swiss-Prot accession number, P00810) of the purified protein indicated that IRT-4 differed from TEM-1 by two substitutions: Leu for Met-69 (ABL numbering) and Asp for Asn-276. A Met-69-Leu variant of TEM-1, obtained by site-directed mutagenesis, has been described as resistant to clavulanate. The Asp for Asn-276 substitution has not been reported previously. The side chains of Asp-276 and Arg-244 were expected to interact. Determinations of 50% inhibitory concentrations of beta-lactamase inhibitors and substrate profile of IRT-4 suggested that such an ionic bond was implicated in the alteration of the mechanistic process of TEM-1 beta-lactamase.

Cloning and expression of the penicillinase from a borderline methicillin-susceptible Staphylococcus aureus strain in Escherichia coli.

The blaZ gene contained in a single 17.2-kb beta-lactamase plasmid from a borderline methicillin-susceptible Staphylococcus aureus strain (a53) has been cloned in Escherichia coli. A Bluescript II derivative in which the ampicillin resistance gene has been replaced with the chloramphenicol resistance gene was used as a multi-copy vector. One ampicillin-resistant colony was detected among 31 chloramphenicol-resistant transformants selected. This E. coli clone harbored a recombinant plasmid (pAH12) containing two different staphylococcal HindIII inserts (7.0 and 5.3 kb), of which only the former hybridized with a blaZ probe. The clone showed an ampicillin MIC of > 1024 micrograms ml-1, independently of the inoculum size used, and produced large amounts of beta-lactamase, which hydrolyzed nitrocefin and penicillin G but not methicillin of the beta-lactamase substrate, padac. In contrast, S. aureus a53 hydrolyzed all four substrates. The fact that high levels of staphylococcal penicillinase are unable to cause methicillin hydrolysis confirms that penicillinase hyperproduction is unlikely to be the true mechanism responsible for the borderline phenotype. These results also suggest that the two different beta-lactamases (penicillinase and methicillinase) associated with borderline S. aureus strains have a different genetic origin.

Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of β‐lactamase

In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.

Interaction of β-lactamases I and II from Bacillus cereus with semisynthetic cephamycins. Kinetic studies

The influence of C-6 alpha- or C-7 alpha-methoxylation of the beta-lactam ring in the catalytic action of class A and B beta-lactamases has been investigated. For this purpose the kinetic behaviour of beta-lactamases I (class A) and II (class B) from Bacillus cereus was analysed by using several cephamycins, moxalactam, temocillin and related antibiotics. These compounds behaved as poor substrates for beta-lactamase II, with high Km values and very low catalytic efficiencies. In the case of beta-lactamase I, the substitution of a methoxy group for a H atom at C-7 alpha or C-6 alpha decreased the affinity of the substrates for the enzyme. Furthermore, the acylation of cephamycins was completely blocked, whereas that of penicillins was slowed down by a factor of 10(4)-10(5), acylation being the rate-determining step of the process.

N-(Phenylacetyl)glycyl-D-aziridine-2-carboxylate, an acylic amide substrate of .beta.-lactamases: importance of the shape of the substrate in .beta.-lactamase evolution

Certain acyclic depsipeptides, but not peptides, are substrates of typical beta-lactamases [Pratt, R.F., & Govardhan, C.P. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1302]. This may reflect either the greater chemical reactivity of depsipeptides (and of beta-lactams, the natural substrates) than peptides or the greater ease of distortion of the depsipeptide (ester) than the peptide (amide) group into a penicillin-like conformation. The latter explanation has been shown to be more likely by employment of a novel beta-lactamase substrate. N-(phenylacetyl)glycyl-D-aziridine-2-carboxylate, which combines a high chemical reactivity with a close to tetrahedral amide nitrogen atom. Although this substrate was better (higher kcat/KM) than a comparable depsipeptide for beta-lactamases, it was poorer than the depsipeptide for the Streptomyces R61 D-alanyl-D-alanine peptidase (which catalyzes specific peptide hydrolysis). It therefore seems likely that one vital feature of the putative evolution of a DD-peptidase into a beta-lactamase would have been modification of the active site to, on one hand, accommodate bicyclic beta-lactams and, on the other, exclude productive binding of planar acyclic amides. Certain serine beta-lactamases and the R61 DD-peptidase also catalyze methanolysis and aminolysis by D-phenylalanine of the N-acylaziridine. The latter reaction, the first amide aminolysis shown to be catalyzed by a beta-lactamase, is a very close analogue of the transpeptidase reaction of DD-peptidases. The methanolysis reaction appeared to proceed by way of the same acyl-enzyme intermediate as formed from depsipeptides possessing the same acyl moiety as the aziridine. The kinetics of methanolysis were employed to determine whether acylation or deacylation was rate limiting to the hydrolysis reaction under saturating substrate concentrations. The kinetics of the aminolysis reaction, catalyzed by the Enterobacter cloacae P99 beta-lactamase, showed the characteristics of, and were interpreted in terms of, a sequential mechanism previously deduced for depsipeptides and this enzyme [Pazhanisamy, S., & Pratt, R. F. (1989) Biochemistry 28, 6875-6882]. This mechanism features two separate binding sites, only one of which is productive. Strikingly, the binding of the N-acylaziridine to the nonproductive site was very tight, such that essentially all hydrolysis at substrate concentrations above 0.1Km proceeded via the ternary complex; this could also be true of penicillins.

Antimicrobial activity and beta-lactamase stability of BMY-28232, parent compound of an oral cephalosporin

BMY-28232, an aminothiazolyl imino methoxy cephalosporin which is available as an orally absorbed acetoxyethyl ester, inhibited strains of methicillin-susceptible Staphylococcus aureus, hemolytic streptococci, Streptococcus pneumoniae, Escherichia coli, Klebsiella species, and many strains of Proteus and Providencia stuartii at concentrations less than 1 microgram/ml, including isolates resistant to cephalexin and cefaclor. It had activity similar to that of cefixime, but was more active against methicillin-susceptible staphylococci. BMY-28232 was a poor substrate for beta-lactamases but was destroyed by the new TEM-3 enzyme, and had less activity against Enterobacter species, Citrobacter freundii, and Proteus vulgaris isolates. Methicillin-resistant staphylococci, Pseudomonas species, enterococci, Listeria monocytogenes, Corynebacterium jeikeium, Bacteroides fragilus and some strains of Clostridium species were resistant to BMY-28232.