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.     

Characterization of β-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry

Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.     

Pollutant-induced modulation in conformation and β-lactamase activity of human serum albumin

Structural changes in human serum albumin (HSA) induced by the pollutants 1-naphthol, 2-naphthol and 8-quinolinol were analyzed by circular dichroism, fluorescence spectroscopy and dynamic light scattering. The alteration in protein conformational stability was determined by helical content induction (from 55 to 75%) upon protein-pollutant interactions. Domain plasticity is responsible for the temperature-mediated unfolding of HSA. These findings were compared to HSA-hydrolase activity. We found that though HSA is a monomeric protein, it shows heterotropic allostericity for β-lactamase activity in the presence of pollutants, which act as K- and V-type non-essential activators. Pollutants cause conformational changes and catalytic modifications of the protein (increase in β-lactamase activity from 100 to 200%). HSA-pollutant interactions mediate other protein-ligand interactions, such as HSA-nitrocefin. Therefore, this protein can exist in different conformations with different catalytic properties depending on activator binding. This is the first report to demonstrate the catalytic allostericity of HSA through a mechanistic approach. We also show a correlation with non-microbial drug resistance as HSA is capable of self-hydrolysis of β-lactam drugs, which is further potentiated by pollutants due to conformational changes in HSA.     

Characterization of β-lactamase activity using isothermal titration calorimetry

BackgroundHydrolysis of β-lactam antibiotic by β-lactamase is the most common mechanism of β-lactam resistance in clinical isolates. Timely detection and characterization of β-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on β-lactamases.MethodsA new assay was developed for the study of β-lactamase activity in protein solutions (Metallo-β-lactamase L1) and in clinical bacterial cells, based on heat-flow changes derived from enzymatic hydrolysis of β-lactams using isothermal titration calorimetry.Results(1) The thermokinetic parameters of three antibiotics (penicillin G, cefazolin and imipenem) and the inhibition constant of an azolylthioacetamide inhibitor were determined using the calorimetric assay. The results from the calorimetric assays were consistent with the data from the spectrophotometric assay. (2) The values of heat change in the calorimetric assay using two clinical Escherichia coli strains correlated well with their antibiotic susceptibility results from the broth dilution experiment. The subtypes of β-lactamase were also determined in the calorimetric assay.ConclusionsThe ITC assay is a reliable and fast method to study β-lactamase enzyme kinetics and inhibition. It can also provide thermodynamic information on antibiotic hydrolysis, which has been taken advantage of in this work to study β-lactamase activity in two clinical Escherichia coli isolates.General significanceAs the first calorimetric study of β-lactamase activity, it may provide a new assay to assist biomedical validation of new β-lactamase inhibitors, and also has potential applications on rapid antibiotic susceptibility testing and screening β-lactamase producing bacteria.     

Circular permutation in the Ω-loop of TEM-1 β-lactamase results in improved activity and altered substrate specificity

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein.     

Promiscuous (+)-γ-lactamase activity of an amidase from nitrile hydratase pathway for efficient synthesis of carbocyclic nucleosides intermediate

Based on bioinformatics analysis, the promiscuous (+)-γ-lactamase activity of an amidase was identified in Rhodococcus erythropolis PR4 and found to be involved in the nitrile hydratase pathway. The amidase is highly enantioselective and can be used in the kinetic resolution of the Vince lactam. The known structure provides a rare insight into the catalytic mechanism of (+)-γ-lactamase with absolute chiral selectivity. This lactamase was cloned, purified, biochemically characterized, and demonstrated to be an ideal catalyst for the preparation of carbocyclic nucleosides of pharmaceutical interest. The chiral selectivity of this enzyme was investigated by molecular docking and site-specific mutagenesis, which provides a foundation for further engineering of these versatile biocatalysts.     

Characterization of a recombinant (?)γ-lactamase from Microbacterium hydrocarbonoxydans

A (−)γ-lactamase, Mhg, from Microbacterium hydrocarbonoxydans was over-expressed in E. coli and was characterized after purification. The maximum activity was at pH 8.0 and 60°C and the half life of Mhg was ~30 min at 75°C. The enzyme was activated by DTT. The catalytic triad of the (−)γ-lactamase is comprised of residues Ser98, Asp230, and His259 and an oxyanion hole was formed by Tyr32 and Met99 according to the alignment results. Under native conditions, the (−)γ-lactamase consists of two 31 kDa homodimers.     

Phosphonic acid analogue of forfenicine — synthesis, antibacterial activity and degradation byPseudomonas fluorescens

Summary Phosphonic acid analogue of forfenicine, amino (p-formylbenzyl)-phosphonic acid, was synthesized and evaluated as antibacterial agent. As indicated by disc diffusion test this compound was found to inhibit significantly the growth ofBacillus subtilis andStaphylococcus aureus and moderately the growth ofEscherichia coli. Resistance ofPseudomonas fluorescens to the action of the aminophosphonate may result from the ability of the strain to degrade this compound.     

γ-Glutamyltranspeptidase in echinoderm eggs and larvae: fertilization-dependent and developmentally-induced changes in specific activity

• 1.1. γ-Glutamyltranspeptidase is present in echinoderm eggs and larvae: in homogenates the level of activity is comparable to that of rat cerebral cortex.
• 2.2. In eggs of Lytechinus pictus, fertilization induces an early rapid and sustained (5 min–6 hr) 37% increase in the activity of γ-glutamyltranspeptidase in homogenate fractions.
• 3.3. Relative to these homogenate levels, the specific activity of γ-glutamyltranspeptidase are ≈60% lower in 40,000 g supernatant fractions and 2.7-fold higher in 40,000 g particulate fractions in both unfertilized and 15 min post-fertilized Lytechinus pictus eggs.
• 4.4. The subcellular distribution of γ-glutamyltranspeptidase is the same in both unfertilized and 15-min post-fertilized Lytechinus pictus eggs: 78% in 40,000 g particulate fractions, 22% in 40,000 g soluble fractions.
• 5.5. In both unfertilized and 15 min post-fertilized eggs of Lytechinus pictus the enzyme responds to heat (50 vs 37°C) by activation in a similar manner: 1.72- and 1.68-fold homogenates; 2.6- and 3.0-fold in supernatants; 1.97- and 1.90-fold in particulate fractions.
• 6.6. In homogenates of Pisaster ochraceous larvae, γ-glutamyltranspeptidase activity increases steadily during the course of larval development: relative to the low activity at day 5, activities exhibit an increase of 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28, respectively.

     

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.     

Induction of alkane-oxidizing andα-olefin-epoxidizing enzymes by a non-hydrocarbon in aPseudomonas

A strain ofPseudomonas aeruginosa could be induced to oxidizen-paraffins and to epoxidize-olefins by treating peptone-grown cells with 1,6-hexanediol or by growing them on this substrate. Of some related alcohols and acids investigated, only a few showed weak inducing capacities.Shell Research N.V.     

Dengue virus infection-enhancing activity in serum samples with neutralizing activity as determined by using FcγR-expressing cells

Background

Progress in dengue vaccine development has been hampered by limited understanding of protective immunity against dengue virus infection. Conventional neutralizing antibody titration assays that use FcγR-negative cells do not consider possible infection-enhancement activity. We reasoned that as FcγR-expressing cells are the major target cells of dengue virus, neutralizing antibody titration assays using FcγR-expressing cells that determine the sum of neutralizing and infection-enhancing activity, may better reflect the biological properties of antibodies in vivo.

Methods and Findings

We evaluated serum samples from 80 residents of a dengue endemic country, Malaysia, for neutralizing activity, and infection-enhancing activity at 1∶10 serum dilution by using FcγR-negative BHK cells and FcγR-expressing BHK cells. The serum samples consisted of a panel of patients with acute DENV infection (31%, 25/80) and a panel of donors without acute DENV infection (69%, 55/80). A high proportion of the tested serum samples (75%, 60/80) demonstrated DENV neutralizing activity (PRNT₅₀≥10) and infection-enhancing activity. Eleven of 18 serum samples from patients with acute secondary DENV infection demonstrated neutralizing activity to the infecting serotype determined by using FcγR-negative BHK cells (PRNT₅₀≥10), but not when determined by using FcγR-expressing cells.

Conclusion

Human serum samples with low neutralizing activity determined by using FcγR-negative cells showed DENV infection-enhancing activity using FcγR-expressing cells, whereas those with high neutralizing activity determined by using FcγR-negative cells demonstrate low or no infection-enhancing activity using FcγR-expressing cells. The results suggest an inverse relationship between neutralizing antibody titer and infection-enhancing activity, and that neutralizing activity determined by using FcγR-expressing cells, and not the activity determined by using FcγR-negative cells, may better reflect protection to DENV infection in vivo.     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Fluorescent TEM-1 β-lactamase with wild-type activity as a rapid drug sensor for in?vitro drug screening

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 β-lactamase through the combined strategy of Val²¹⁶→Cys²¹⁶ mutation and fluorophore labelling for in vitro drug screening. The Val²¹⁶ residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys²¹⁶ residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when β-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent β-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor''s role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val²¹⁶→Cys²¹⁶ mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type β-lactamase variants for in vitro drug screening.     

3-Oxo-γ-costic acid fungal-transformation generates eudesmane sesquiterpenes with in vitro tumor-inhibitory activity

While select eudesmane sesquiterpenes exhibit anti-neoplastic activity, tumor-inhibition for costic-acids has not been established. Here biological activity of 3-oxo-γ-costic acid (1), previously isolated from Chiliadenus montanus, as well as new sesquiterpenes (2–5) and the known derivative, 3-oxoeudesma-1,4,11(13)-trien-7-1061αH-l2-oic acid (6), all produced from 1 by the fungus Athelia rolfsii, are reported. Structures were elucidated using MS and NMR spectroscopy with activity-screening utilizing human colon- and lung-tumor lines, Caco-2 and A549 respectively. Compound 1 exhibited anti-proliferative activity against Caco-2 (IC₅₀ 39 µM) and 2 was active against A549 (IC₅₀ 74 µM) suggesting therapeutic potential for the original substrate and a bio-transformed product.     

Synthesis and biological activity of N-aroyl-tetrahydro-γ-carbolines

Research on dual inhibitors of both 5-LOX and COXs gained interest due to the overexpressions of these enzymes during the malignant state of the evolution of prostate cancer. In order to take part in this research, new N-aroyl-tetrahydro-γ-carbolines issued from the modification of Indomethacin have been synthesised. As for the NSAIDs, the compounds have been tested for their activity against COX₁, COX₂ plus against 5-LOX and against the proliferation of malignant prostate cancer. Interesting cytotoxic activities and selectivities of some tetrahydro-γ-carboline derivatives have been obtained.     

Differential activity of rosiglitazone enantiomers at PPARγ

Analysis of the enantiomers of rosiglitazone in a PPARγ binding assay suggests that the (S)-(−)-isomer is responsible for the antidiabetic activity.     

Substituted aryl malonamates as new serine β-lactamase substrates: Structure–activity studies

A series of substituted aryl malonamates have been prepared. These compounds are analogues of aryl phenaceturates where the amido side chain has been replaced by a retro-amide. Like the phenaceturates, these compounds are substrates of typical class A and class C β-lactamases, particularly of the latter, and of soluble DD-peptidases. The effect of substituents α to the ester carbonyl group on turnover by these enzymes is similar to that in the phenaceturates. On the other hand, N-alkylation of the side chain amide of malonamates, but not of phenaceturates, retains the susceptibility of the compounds to hydrolysis by β-lactamases. This reactivity is not enhanced, however, by bridging the amide nitrogen and Cα atoms. A phosphonate analogue of the malonamates was found to be an irreversible inhibitor of the β-lactamases. These results, therefore, provide further evidence for the covalent access of compounds bearing retro-amide side chains to the active sites of β-lactam-recognizing enzymes.     

Is ethylene involved in regulation of root ferric reductase activity of dicotyledonous species under iron deficiency?

Most dicotyledonous species respond to Fe deficiency by developing some mechanisms known as Fe-deficiency responses. The role of ethylene in regulation of root ferric reductase activity of wild-type tomato (Lycopersicon esculentum L.) and its mutant Never ripe (Nr), bean (Phaseolus vulgaris L., cv. Bifeng 80-30), and cucumber (Cucumis sativus L., cv. Xintaimici) plants grown in nutrient solution without Fe supply was studied under controlled condition. The results show that: (i) the tomato mutant Nr, which is insensitive to ethylene, presented rapid increase in root ferric reductase activity after omitting Fe from the nutrient solution; (ii) the initial time for increase in root ferric reductase activity was earlier than that in ethylene production after onset of Fe deficiency in the three species; (iii) like cobalt (3 μM Co²⁺), an inhibitor for ethylene production, high concentration of zinc (50 μM Zn²⁺) and copper (5 μM Cu²⁺) also suppressed the increase in root ferric reductase activity of Fe-starved plants; (iv) under Fe-sufficient conditions, indol-3-butylric acid (IBA) stimulated root ferric reductase activity of cucumber and bean plants, and this stimulating effect could not be suppressed by aminoethoxyvinylglycine (AVG, an inhibitor for ethylene synthesis). These results suggested that ethylene might not be directly involved in the regulation of root ferric reductase activity of Fe-deficient dicotyledonous species.