Extracellular β-lactamase from Escherichia coli

Summary Escherichia coli strain O 127: K63: (B8): H—was grown in nutrient broth (Difco). Penicillinase activity was found in the culture supernatant after only five hours of incubation, i.e. during the exponential phase of growth. At this phase the levels of typical intracellular markers, did not indicate cell lysis or gross cell damage. The bioautographic revelation of penicillin-splitting enzymes on electropherograms of cell-free liquids confirmed the presence of one basic broad-spectrum -lactamase. This possibly extracellular -lactamase seems to be also present in the cellular extracts where it coexists with several other cell-bound penicillinases.     

Metal cofactor requirement of β-lactamase II

1. The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn(2+)-containing enzyme. 2. Removal of Zn(2+) led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn(2+) by Co(2+), Cd(2+), Mn(2+) or Hg(2+) gave enzymes with significant, but lower, beta-lactamase activity. No activity was detected in the presence of Cu(2+), Ni(2+), Mg(2+) or Ca(2+). 4. Equilibrium dialysis indicated that the enzyme had at least two Zn(2+) binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn(2+) indicated that activity paralleled binding of Zn(2+) to the site of highest affinity. 5. Replacement of Zn(2+) by Co(2+) and Cd(2+) gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured beta-lactamase II with Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce beta-lactamase II activity in high yield.     

Human-associated extended-spectrum β-lactamase in the Antarctic

Escherichia coli bacteria with extended-spectrum β-lactamase (ESBL) type CTX-M resistance were isolated from water samples collected close to research stations in Antarctica. The isolates had bla(CTX-M-1) and bla(CTX-M-15) genotypes and sequence types (ST) indicative of a human-associated origin. This is the first record of ESBL-producing enterobacteria from Antarctica.     

Identification of a β-lactamase inhibitory protein variant that is a potent inhibitor of Staphylococcus PC1 β-lactamase

β-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits the treatment strategies for Staphylococcus infections. The goals of this study were to determine the binding affinity of BLIP for Staphylococcus aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (K_i) for PC1 β-lactamase was measured at 350 nM, and isothermal titration calorimetry experiments indicated a binding constant (K_d) of 380 nM. Twenty-three residue positions in BLIP that contact β-lactamase were randomized, and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP_K74G mutant was the dominant clone selected, and it was found to inhibit the PC1 β-lactamase with a K_i of 42 nM, while calorimetry indicated a K_d of 26 nM. Molecular modeling studies suggested that BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme, where it forms a salt bridge with the BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the PC1_A104E enzyme binds BLIP with 15-fold greater affinity than wild-type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with variation in affinity due to altered dissociation rate constants, suggesting that changes in short-range interactions are responsible for the altered binding properties of the mutants.     

Inhibition of R-factor-mediated β-lactamase by Proteus mirabilis

In cultures of Proteus mirabilis the level of R-factor-TEM-mediated -lactamase activity rose during the logarithmic phase, reached a maximum value at the end of this phase and fell rapidly when the cells entered the stationary phase. Supernatants from cultures in the stationary phase were found to inhibit R-TEM -lactamase activity both in cell-free preparations and in intact cells. The inhibitory activity may be due to a proteolytic enzyme excreted into the medium.     

Fluorogenic cephalosporin substrates for β-lactamase TEM-1

Cephalosporin was used to synthesize soluble and precipitating fluorogenic β-lactam substrates that demonstrated differential catalytic hydrolysis by three different subtypes of β-lactamase: TEM-1 (class A), p99 (class C), and a Bacillus cereus enzyme sold by Genzyme (class B). The most successful soluble substrate contained difluorofluorescein (Oregon Green 488) ligated to two cephalosporin moieties that, therefore, required two turnovers to produce the fluorescent Oregon Green 488 leaving group. The bis-cephalosporin modification was required so that the final reaction product was the Oregon Green 488 carboxylic acid rather than a less bright phenolic adduct of the dye. Hydrolysis in pH 5.5 Mes and pH 7.2 phosphate-buffered saline (PBS) buffers was similar, but in pH 8.0 Tris the hydrolysis rate nearly doubled. Activity of the β-lactamases on the various substrates was shown to depend highly on the linker between the cephalosporin and the fluorophore, with an allyl linker promoting faster turnover than a phenol ether linker. Measured K_m values for dichlorofluorescein and difluorofluorescein cephalosporin substrates were approximately the same as K_m values for penicillin G and ampicillin found in the literature (∼30–40 μM).     

Separation, purification and properties of β-lactamase I and β-lactamase II from Bacillus cereus 569/H/9

1. A procedure was devised which is suitable for the isolation of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9 on a large scale. After adsorption on to Celite both enzymes were eluted in good yield and separated by chromatography on Sephadex CM-50. 2. beta-Lactamase I was separated into three main components by isoelectric focusing and into two components by chromatography. 3. The Zn(2+)-requiring beta-lactamase II obtained by this procedure had a lower molecular weight (22000) than beta-lactamase I (28000) and also differed from the latter in containing one cysteine residue. 4. The beta-lactamase II contained no carbohydrate, but showed the thermostability of the enzyme isolated earlier as a protein-carbohydrate complex. 5. Amino acid analyses and tryptic-digest ;maps' indicate that some degree of homology between beta-lactamase I and beta-lactamase II is possible, but that beta-lactamase I is not composed of the entire sequence of beta-lactamase II together with an additional peptide fragment. 6. A 6-methylpenicillin and a 7-methylcephalosporin showed much lower affinities for both enzymes than did penicillins and cephalosporins themselves.     

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.     

The dimeric nature of an R-factor mediated β-lactamase

The molecular weight of the β-lactamase mediated by R46 as determined by SDS-gel electrophoresis is about half that of the native enzyme, indicating the presence of subunits. Similar evidence for subunits was found with the β-lactamase mediated by R55. $\text{In}\text{vivo}$ hybridization occurred between the R46 and R55 β-lactamases.     

A chromogenic cephalosporin for β-lactamase inhibitor screening assays

Production of β-lactamases is the primary mechanism of antibiotic resistance employed by gram-negative pathogens. Chromogenic β-lactams are important reagents for detection and assay of β-lactamases, but limited commercial availability and exorbitant pricing of these compounds are prohibitive. Here we describe a straightforward synthesis of a chromogenic cephalosporin for β-lactamase assay that gives an overall yield of 74%. On hydrolysis, its λ(max) undergoes a bathochromic shift that is easy to see and measure spectrophotometrically with a Δε(442 nm) of 14,500cm(-1)M(-1). This compound was shown to be a substrate for a variety of β-lactamases.     

Extracellular location of a β-lactamase produced by Acinetobacter calcoaceticus

Summary Acinetobacter calcoaceticus strains 69 V and CCM 5593 formed a -lactamase that was predominantly found extracellularly in the culture medium. The enzyme has the characteristics of an inducible chromosomally mediated cephalosporinase. Neither cell lysis nor periplasmic leakage are responsible for the extracellular location. The mechanism of secretion is not known.Offprint requests to: P. Borneleit     

Serendipitous discovery of aryl boronic acids as β-lactamase inhibitors

High throughput screening for β-lactamase inhibitors afforded biphenyl hits such as 1. Hit confirmation and X-ray soaking experiments with Pseudomonas Aeruginosa AmpC enzyme led to the identification of an aryl boronic acid-serine complex 4, which was formed from phenyl boronic acid 8 (an impurity in compound 1) and ethylene glycol (the cryoprotectant in the soaking experiment).     

The evolution of cefotaximase activity in the TEM β-lactamase

The development of a molecular-level understanding of drug resistance through β-lactamase is critical not only in designing newer-generation antibacterial agents but also in providing insight into the evolutionary mechanisms of enzymes in general. In the present study, we have evaluated the effect of four drug resistance mutations (A42G, E104K, G238S, and M182T) on the cefotaximase activity of the TEM-1 β-lactamase. Using computational methods, including docking and molecular mechanics calculations, we have been able to correctly identify the relative order of catalytic activities associated with these four single point mutants. Further analyses suggest that the changes in catalytic efficiency for mutant enzymes are correlated to structural changes within the binding site. Based on the energetic and structural analyses of the wild-type and mutant enzymes, structural rearrangement is suggested as a mechanism of evolution of drug resistance through TEM β-lactamase. The present study not only provides molecular-level insight into the effect of four drug resistance mutations on the structure and function of the TEM β-lactamase but also establishes a foundation for a future molecular-level analysis of complete evolutionary trajectory for this class of enzymes.     

Serendipitous discovery of α-hydroxyalkyl esters as β-lactamase substrates

O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both d- and l-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and d-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.     

Inactivation of exogenous β-lactamase in transformed yeast Saccharomyces cerevisiae

Summary The stability of bacterial -lactamase in transformedSacharomyces cerevisiae grown on glucose was studied. A culture of a prototrophic strain showed marked inactivation shortly before the stationary phase. This was also observed in cells starved of nitrogen. The level of reserve carbohydrate was lower both in the stationary-phase culture of the auxotroph and in the glucose-starved culture of the prototroph, where less inactivation was observed. Such a close correlation suggests that inactivation may be triggered mainly in response to nitrogen-limitation which regulates reserve carbohydrate metabolism.     

Boronic acid inhibitors of the class A β-lactamase KPC-2

The rapid rise of antimicrobial resistance is one of the greatest challenges currently facing medical science. The most common cause of resistance to β-lactam antibiotics is the expression of β-lactamase enzymes, such as KPC-2. As such the development of novel inhibitors of KPC-2 and related enzymes is of the upmost importance. We report the design and synthesis of novel boronic acid transition state analogs containing a 1,4-substituted 1,2,3-triazole linker based on the known inhibitor 3-nitrophenyl boronic acid and demonstrate that they are promising scaffolds for the development inhibitors of KPC-2 with the ability to recover sensitivity to the antibiotic cefotaxime.     

Permeation through the cell membrane of a boron-based β-lactamase inhibitor

Bacteria express beta-lactamases to counteract the beneficial action of antibiotics. Benzo[b]-thiophene-2-boronic acid (BZB) derivatives are β-lactamase inhibitors and, as such, promising compounds to be associated with β-lactam antibacterial therapies. The uncharged form of BZB, in particular, is suggested to diffuse through the outer membrane of gram negative bacteria. In this study, through the combination of electrophysiological experiments across reconstituted PC/n-decane bilayers and metadynamics-based free energy calculations, we investigate the permeation mechanism of boronic compounds. Our experimental data establish that BZB passes through the membrane, while computer simulations provide hints for the existence of an aqueous, water-filled monomolecular channel. These findings provide new perspectives for the design of boronic acid derivatives with high membrane permeability.     

Design and synthesis of a β-lactamase activated 5-fluorouracil prodrug

An efficient synthesis of a 5-fluorouracil-cephalosporin prodrug is described for use against colorectal and other cancers in antibody and gene-directed therapies. The compound shows stability in aqueous media until specifically activated by β-lactamase (βL). The kinetic parameters of the 5-fluorouracil-cephalosporin conjugate were determined in the presence of Enterobacter cloacae P99 βL (ECl βL) revealing a K_m = 95.4 μM and V_max = 3.21 μMol min^?1 mg^?1. The data compare favorably to related systems that have been reported and enable testing of this prodrug against cancer cell lines in vitro and in vivo.