Crystal structures of the molecular class A ß-lactamase TEM-171 and its complexes with tazobactam.

The resistance of bacteria to ß-lactam antibiotics is primarily caused by the production of ß-lactamases. Here, novel crystal structures of the native ß-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical ß-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published ß-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new ß-lactamase inhibitors.

Isolation and Characterization of Ochrobactrum tritici for Penicillin V Potassium Degradation.

Substantial concentrations of penicillin V potassium (PVK) have been found in livestock manure, soil, and wastewater effluents, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, bacterial strains capable of degrading PVK were isolated from sludge and characterized. Strain X-2 was selected for biodegradation of PVK. Based on morphological observations and 16S rRNA gene sequencing, strain X-2 was identified as an Ochrobactrum tritici strain. To enhance the PVK degradation ability of PVK, a whole-cell biodegradation process of Ochrobactrum tritici X-2 was established and optimized. In the whole-cell biodegradation process, the optimal temperature and pH were 30°C and 7.0, respectively. Under the optimized conditions, the degradation rate using 0.5 mg/ml PVK reached 100% within 3 h. During biodegradation, two major metabolites were detected: penicilloic acid and phenolic acid. The present study provides a novel method for the biodegradation of PVK using Ochrobactrum tritici strains, which represent promising candidates for the industrial biodegradation of PVK.IMPORTANCE Substantial concentrations of penicillin V potassium (PVK) have been found in the environment, which may pose potential threats to human health and contribute to the emergence of penicillin-resistant bacterial strains. In this study, antibiotic-degrading bacterial strains for PVK were isolated from sludge and characterized. Ochrobactrum tritici was selected for the biodegradation of PVK with high efficiency. To enhance its PVK degradation ability, a whole-cell biodegradation process was established and optimized using Ochrobactrum tritici The degradation rate with 0.5 mg/ml PVK reached 100% within 3 h. The potential biodegradation pathway was also investigated. To the best of our knowledge, the present study provides new insights into the biodegradation of PVK using an Ochrobactrum tritici strain, a promising candidate strain for the industrial biodegradation of ß-lactam antibiotics.

Directed evolution of a penicillin V acylase from Bacillus sphaericus to improve its catalytic efficiency for 6-APA production.

Penicillin acylase is commonly used to produce the medical intermediates of 6-Aminopenicillanic acid (6-APA) and 7-Aminodesacetoxycephalosporanic acid (7-ADCA) in industrial process. Nowadays, Penicillin G acylase (PGA) has been widely applied for making pharmaceutical intermediates, while penicillin V acylase (PVA) has been less used for that due to its low activity and poor conversion. In this study, a PVA from Bacillus sphaericus (BspPVA) was employed for directed evolution study with hoping to increase its catalytic efficiency. Finally, a triple mutant BspPVA-3 (T63S/N198Y/S110C) was obtained with 12.4-fold specific activity and 11.3-fold catalytic efficiency higher than BspPVA-wt (wild type of BspPVA). Moreover, the conversion yields of 6-APA catalyzed by BspPVA-3 reached 98% with 20% (w/v) penicillin V as substrate, which was significantly higher than that of the BspPVA-wt (85%). Based on the analysis of modeling, the enhancement of specific activity of mutant BspPVA-3 was probably attributed to the changes in the number of hydrogen bonds within the molecules. The triple mutant PVA developed in this study has a potential for large-scale industrial application for 6-APA production.

Mechanisms of proton relay and product release by Class A ß-lactamase at ultrahigh resolution.

The ß-lactam antibiotics inhibit penicillin-binding proteins (PBPs) by forming a stable, covalent, acyl-enzyme complex. During the evolution from PBPs to Class A ß-lactamases, the ß-lactamases acquired Glu166 to activate a catalytic water and cleave the acyl-enzyme bond. Here we present three product complex crystal structures of CTX-M-14 Class A ß-lactamase with a ruthenocene-conjugated penicillin-a 0.85 Å resolution structure of E166A mutant complexed with the penilloate product, a 1.30 Å resolution complex structure of the same mutant with the penicilloate product, and a 1.18 Å resolution complex structure of S70G mutant with a penicilloate product epimer-shedding light on the catalytic mechanisms and product inhibition of PBPs and Class A ß-lactamases. The E166A-penilloate complex captured the hydrogen bonding network following the protonation of the leaving group and, for the first time, unambiguously show that the ring nitrogen donates a proton to Ser130, which in turn donates a proton to Lys73. These observations indicate that in the absence of Glu166, the equivalent lysine would be neutral in PBPs and therefore capable of serving as the general base to activate the catalytic serine. Together with previous results, this structure suggests a common proton relay network shared by Class A ß-lactamases and PBPs, from the catalytic serine to the lysine, and ultimately to the ring nitrogen. Additionally, the E166A-penicilloate complex reveals previously unseen conformational changes of key catalytic residues during the release of the product, and is the first structure to capture the hydrolyzed product in the presence of an unmutated catalytic serine. DATABASE: Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.

Pharmacokinetics of flucloxacillin and its metabolites in patients with renal failure: Impact on liver toxicity
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OBJECTIVE: The antimicrobial agent flucloxacillin is a potential cause of drug-induced liver disease, but the underlying mechanisms for toxicity have not been fully elucidated. As in-vitro and in-vivo findings suggest that biotransformation products contribute to hepatotoxicity, the purpose of this study was to characterize formation and accumulation of its metabolites in patients with renal failure. METHODS: Twelve intensive care patients undergoing continuous venovenous hemofiltration received 4.0 g flucloxacillin as single and repeated infusion. Blood and dialysate samples were collected and analyzed for flucloxacillin and its metabolites by HPLC. RESULTS: The overall amounts of the flucloxacillin metabolites 5'-hydroxymethylflucloxacillin (5-OH-FX), 5'-hydroxymethylflucloxacillin-penicilloic acid (5-OH-PA), and flucloxacillin-penicilloic acid (FX-PA) produced varied considerably between patients, and accounted for 3.62 - 35.9% of total flucloxacillin concentration (flucloxacillin + metabolites) in the plasma. Clearance rates and sieving coefficients for 5-OH-FX and FX-PA were comparable to that of the parent drug, although removal of 5-OH-PA was decreased. Using an isolated perfused rat liver model we demonstrated that 5-OH-FX reached concentrations in the bile (240.5 ± 84.2 nmoles/mL) that were sufficient to exert cytotoxic effects, unlike either of the two penicilloic acids. CONCLUSIONS: Based on data from perfused rat livers, high biliary concentrations of 5-OH-FX might also be observed in our patients explaining why LDH, bilirubin, and alkaline phosphatase were elevated in up to 8/12 patients after repeated infusion of flucloxacillin. Liver toxicity of flucloxacillin might therefore be observed in patients with renal impairment after continuously elevated 5-OH-FX levels.
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Targeted Metabolomics Analysis Identifies Intestinal Microbiota-Derived Urinary Biomarkers of Colonization Resistance in Antibiotic-Treated Mice.

Antibiotics excreted into the intestinal tract may disrupt the microbiota that provide colonization resistance against enteric pathogens and alter normal metabolic functions of the microbiota. Many of the bacterial metabolites produced in the intestinal tract are absorbed systemically and excreted in urine. Here, we used a mouse model to test the hypothesis that alterations in levels of targeted bacterial metabolites in urine specimens could provide useful biomarkers indicating disrupted or intact colonization resistance. To assess in vivo colonization resistance, mice were challenged with Clostridium difficile spores orally 3, 6, and 11 days after the completion of 2 days of treatment with piperacillin-tazobactam, aztreonam, or saline. For concurrent groups of antibiotic-treated mice, urine samples were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify the concentrations of 11 compounds targeted as potential biomarkers of colonization resistance. Aztreonam did not affect colonization resistance, whereas piperacillin-tazobactam disrupted colonization resistance 3 days after piperacillin-tazobactam treatment, with complete recovery by 11 days after treatment. Three of the 11 compounds exhibited a statistically significant and >10-fold increase (the tryptophan metabolite N-acetyltryptophan) or decrease (the plant polyphenyl derivatives cinnamoylglycine and enterodiol) in concentrations in urine 3 days after piperacillin-tazobactam treatment, followed by recovery to baseline that coincided with the restoration of in vivo colonization resistance. These urinary metabolites could provide useful and easily accessible biomarkers indicating intact or disrupted colonization resistance during and after antibiotic treatment.

Biotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase.

The production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1 hr at pH 5.0 and 35°C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4°C with minimal loss in activity and were reusable up to 10 cycles with 1-hr hardening in CaCl2 between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.

A breakthrough in enzyme technology to fight penicillin resistance-industrial application of penicillin amidase.

Enzymatic penicillin hydrolysis by penicillin amidase (also penicillin acylase, PA) represents a Landmark: the first industrially and economically highly important process using an immobilized biocatalyst. Resistance of infective bacteria to antibiotics had become a major topic of research and industrial activities. Solutions to this problem, the antibiotics resistance of infective microorganisms, required the search for new antibiotics, but also the development of derivatives, notably penicillin derivatives, that overcame resistance. An obvious route was to hydrolyse penicillin to 6-aminopenicillanic acid (6-APA), as a first step, for the introduction via chemical synthesis of various different side chains. Hydrolysis via chemical reaction sequences was tedious requiring large amounts of toxic chemicals, and they were cost intensive. Enzymatic hydrolysis using penicillin amidase represented a much more elegant route. The basis for such a solution was the development of techniques for enzyme immobilization, a highly difficult task with respect to industrial application. Two pioneer groups started to develop solutions to this problem in the late 1960s and 1970s: that of Günter Schmidt-Kastner at Bayer AG (Germany) and that of Malcolm Lilly of Imperial College London. Here, one example of this development, that at Bayer, will be presented in more detail since it illustrates well the achievement of a solution to the problems of industrial application of enzymatic processes, notably development of an immobilization method for penicillin amidase suitable for scale up to application in industrial reactors under economic conditions. A range of bottlenecks and technical problems of large-scale application had to be overcome. Data giving an inside view of this pioneer achievement in the early phase of the new field of biocatalysis are presented. The development finally resulted in a highly innovative and commercially important enzymatic process to produce 6-APA that created a new antibiotics industry and that opened the way for the establishment of over 100 industrial processes with immobilized biocatalysts worldwide today.

Sub-Inhibitory Concentration of Piperacillin-Tazobactam May be Related to Virulence Properties of Filamentous Escherichia coli.

Sub-inhibitory concentrations of antibiotics are always generated as a consequence of antimicrobial therapy and the effects of such residual products in bacterial morphology are well documented, especially the filamentation generated by beta-lactams. The aim of this study was to investigate some morphological and pathological aspects (virulence factors) of Escherichia coli cultivated under half-minimum inhibitory concentration (1.0 µg/mL) of piperacillin-tazobactam (PTZ sub-MIC). PTZ sub-MIC promoted noticeable changes in the bacterial cells which reach the peak of morphological alterations (filamentation) and complexity at 16 h of antimicrobial exposure. Thereafter the filamentous cells and a control one, not treated with PTZ, were comparatively tested for growth curve; biochemical profile; oxidative stress tolerance; biofilm production and cell hydrophobicity; motility and pathogenicity in vivo. PTZ sub-MIC attenuated the E. coli growth rate, but without changes in carbohydrate fermentation or in traditional biochemical tests. Overall, the treatment of E. coli with sub-MIC of PTZ generated filamentous forms which were accompanied by the inhibition of virulence factors such as the oxidative stress response, biofilm formation, cell surface hydrophobicity, and motility. These results are consistent with the reduced pathogenicity observed for the filamentous E. coli in the murine model of intra-abdominal infection. In other words, the treatment of E. coli with sub-MIC of PTZ suggests a decrease in their virulence.

In vivo kinetic analysis of the penicillin biosynthesis pathway using PAA stimulus response experiments.

In this study we combined experimentation with mathematical modeling to unravel the in vivo kinetic properties of the enzymes and transporters of the penicillin biosynthesis pathway in a high yielding Penicillium chrysogenum strain. The experiment consisted of a step response experiment with the side chain precursor phenyl acetic acid (PAA) in a glucose-limited chemostat. The metabolite data showed that in the absence of PAA all penicillin pathway enzymes were expressed, leading to the production of a significant amount of 6-aminopenicillanic acid (6APA) as end product. After the stepwise perturbation with PAA, the pathway produced PenG within seconds. From the extra- and intracellular metabolite measurements, hypotheses for the secretion mechanisms of penicillin pathway metabolites were derived. A dynamic model of the penicillin biosynthesis pathway was then constructed that included the formation and transport over the cytoplasmic membrane of pathway intermediates, PAA and the product penicillin-G (PenG). The model parameters and changes in the enzyme levels of the penicillin biosynthesis pathway under in vivo conditions were simultaneously estimated using experimental data obtained at three different timescales (seconds, minutes, hours). The model was applied to determine changes in the penicillin pathway enzymes in time, calculate fluxes and analyze the flux control of the pathway. This led to a reassessment of the in vivo behavior of the pathway enzymes and in particular Acyl-CoA:Isopenicillin N Acyltransferase (AT).

Highly sensitive bacterial susceptibility test against penicillin using parylene-matrix chip.

This work presented a highly sensitive bacterial antibiotic susceptibility test through ß-lactamase assay using Parylene-matrix chip. ß-lactamases (EC 3.5.2.6) are an important family of enzymes that confer resistance to ß-lactam antibiotics by catalyzing the hydrolysis of these antibiotics. Here we present a highly sensitive assay to quantitate ß-lactamase-mediated hydrolysis of penicillin into penicilloic acid. Typically, MALDI-TOF mass spectrometry has been used to quantitate low molecular weight analytes and to discriminate them from noise peaks of matrix fragments that occur at low m/z ratios (m/z<500). The ß-lactamase assay for the Escherichia coli antibiotic susceptibility test was carried out using Parylene-matrix chip and MALDI-TOF mass spectrometry. The Parylene-matrix chip was successfully used to quantitate penicillin (m/z: [PEN+H](+)=335.1 and [PEN+Na](+)=357.8) and penicilloic acid (m/z: [PA+H](+)=353.1) in a ß-lactamase assay with minimal interference of low molecular weight noise peaks. The ß-lactamase assay was carried out with an antibiotic-resistant E. coli strain and an antibiotic-susceptible E. coli strain, revealing that the minimum number of E. coli cells required to screen for antibiotic resistance was 1000 cells for the MALDI-TOF mass spectrometry/Parylene-matrix chip assay.

Comparison of the risk of acquiring in vitro resistance to doripenem and tazobactam/piperacillin by CTX-M-15-producing Escherichia coli.

To compare the risk of acquiring in vitro resistance between doripenem and tazobactam/piperacillin by CTX-M-15-producing Escherichia coli, the in vitro frequency of resistance was determined. Four strains carrying multiple ß-lactamases such as blaOXA-1 or blaCTX-M-27 as well as blaCTX-M-15 and blaTEM-1 were used. No resistant colonies appeared on doripenem-containing plates, whereas resistant colonies were obtained from three of four test strains against tazobactam/piperacillin using agar plate containing 8- to 16-fold MIC of each drug. These three acquired tazobactam/piperacillin-resistant strains were not cross-resistant to doripenem, and they showed 1.9- to 3.1-fold higher piperacillin-hydrolysis activity compared to those of each parent strain. The change of each ß-lactamase mRNA expression measured by real-time PCR varied among three resistant strains. One of three tazobactam/piperacillin-resistant strains with less susceptibility to ceftazidime overexpressed both blaCTX-M-15 and blaTEM-1, and the other two strains showed higher mRNA expression of either blaTEM-1 or blaOXA-1. These results demonstrate that multiple ß-lactamases carried by CTX-M-15-producing E. coli contributed to the resistance to tazobactam/piperacillin. On the other hand, these resistant strains maintained susceptibility to doripenem. The risk of acquiring in vitro resistance to doripenem by CTX-M-15-producing E. coli seems to be lower than that to tazobactam/piperacillin.

Interactions of "bora-penicilloates" with serine ß-lactamases and DD-peptidases.

Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial ß-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in ß-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C ß-lactamases but, very unexpectedly, not inhibitors of class A ß-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of ß-lactam antibiotics, where deacylation of ß-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions.

Following drug uptake and reactions inside Escherichia coli cells by Raman microspectroscopy.

Raman microspectroscopy combined with Raman difference spectroscopy reveals the details of chemical reactions within bacterial cells. The method provides direct quantitative data on penetration of druglike molecules into Escherichia coli cells in situ along with the details of drug-target reactions. With this label-free technique, clavulanic acid and tazobactam can be observed as they penetrate into E. coli cells and subsequently inhibit ß-lactamase enzymes produced within these cells. When E. coli cells contain a ß-lactamase that forms a stable complex with an inhibitor, the Raman signature of the known enamine acyl-enzyme complex is detected. From Raman intensities it is facile to measure semiquantitatively the number of clavulanic acid molecules taken up by the lactamase-free cells during growth.

Penicillin G acylase from Achromobacter sp. CCM 4824 : an efficient biocatalyst for syntheses of beta-lactam antibiotics under conditions employed in large-scale processes.

Penicillin G acylase from Achromobacter sp. (NPGA) was studied in the enzymatic synthesis of ß-lactam antibiotics by kinetically controlled N-acylation. When compared with penicillin acylase of Escherichia coli (PGA), the NPGA was significantly more efficient at syntheses of ampicillin and amoxicillin (higher S/H ratio and product accumulation) in the whole range of substrate concentrations. The degree of conversion of 6-aminopenicillanic acid to amoxicillin and ampicillin (160 mM 6-APA, 350 mM acyl donor methylester[Symbol: see text]HCl, pH 6.3, 25 °C, reaction time of 200 min) with immobilized NPGA equaled 96.9 % and 91.1 %, respectively. The enzyme was highly thermostable with maximum activity at 60 °C (pH 8.0) and 65 °C (pH 6.0). Activity half-life at 60 °C (pH 8.0) and at 60 °C (pH 6.0) was 24 min and 6.9 h, respectively. Immobilized NPGA exhibited long operational stability with half-life of about 2,000 cycles for synthesis of amoxicillin at conversion conditions used in large-scale processes (230 mM 6-APA, 340 mM D-4-hydroxyphenylglycine methylester[Symbol: see text]HCl, 27.5 °C, pH 6.25). We discuss our results with literature data available for related penicillin acylases in terms of their industrial potential.

Binding of (5S)-penicilloic acid to penicillin binding protein 3.

ß-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.

Relationship between ceftolozane-tazobactam exposure and drug resistance amplification in a hollow-fiber infection model.

In an era of rapidly emerging antimicrobial-resistant bacteria, it is critical to understand the importance of the relationships among drug exposure, duration of therapy, and selection of drug resistance. Herein we describe the results of studies designed to determine the ceftolozane-tazobactam exposure necessary to prevent the amplification of drug-resistant bacterial subpopulations in a hollow-fiber infection model. The challenge isolate was a CTX-M-15-producing Escherichia coli isolate genetically engineered to transcribe a moderate level of blaCTX-M-15. This organism's blaCTX-M-15 transcription level was confirmed by relative quantitative reverse transcription-PCR (qRT-PCR), ß-lactamase hydrolytic assays, and a ceftolozane MIC value of 16 mg/liter. In these studies, the experimental duration (10 days), ceftolozane-tazobactam dose ratio (2:1), and dosing interval (every 8 h) were selected to approximate those expected to be used clinically. The ceftolozane-tazobactam doses studied ranged from 125-62.5 to 1,500-750 mg. Negative- and positive-control arms included no treatment and piperacillin-tazobactam at 4.5 g every 6 h, respectively. An inverted-U-shaped function best described the relationship between bacterial drug resistance amplification and drug exposure. The least- and most-intensive ceftolozane-tazobactam dosing regimens, i.e., 125-62.5, 750-375, 1,000-500, and 1,500-750 mg, did not amplify drug resistance, while drug resistance amplification was observed with intermediate-intensity dosing regimens (250-125 and 500-250 mg). For the intermediate-intensity ceftolozane-tazobactam dosing regimens, the drug-resistant subpopulation became the dominant population by days 4 to 6. The more-intensive ceftolozane-tazobactam dosing regimens (750-375, 1,000-500, and 1,500-750 mg) not only prevented drug resistance amplification but also virtually sterilized the model system. These data support the selection of ceftolozane-tazobactam dosing regimens that minimize the potential for on-therapy drug resistance amplification.

Why tazobactam and sulbactam have different intermediates population with SHV-1 ß-lactamase: a molecular dynamics study.

The imine intermediates of tazobactam and sulbactam bound to SHV-1 ß-lactamase were investigated by molecular dynamics (MD) simulation respectively. Hydrogen bond networks around active site were found different between tazobactam and sulbactam acyl-enzymes. In tazobactam imine intermediate, it was observed that the triazolyl ring formed stable hydrogen bonds with Asn170 and Thr167. The results suggest that conformation of imine determined the population of intermediates. In imine intermediate of tazobactam, the triazolyl ring is trapped in Thr_Asn pocket, and it restricts the rotation of C5-C6 bond so that tazobactam can only generate trans enamine intermediate. Further, conformational cluster analyses are performed to substantiate the results. These findings provide an explanation for the corresponding experimental results, and will be potentially useful in the development of new inhibitors.

N152G, -S, and -T substitutions in CMY-2 ß-lactamase increase catalytic efficiency for cefoxitin and inactivation rates for tazobactam.

Class C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in the Escherichia coli AmpC and P99 enzymes and have suggested that interactions between C-6' or C-7' substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A ß-lactamase inhibitor with in vitro activity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC50) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C ß-lactamase inhibitors are developed.

Comparative study of 6-APA production by free and agar immobilized bacteria in nutrient broth culture.

In the present study different bacterial samples were isolated from soil of different places of Dibrugarh and screened for biotransformation ability to produce 6-Aminopenicillanic acid. Among ten isolated bacterial samples, three gram positive bacterial samples designated as AKDD-2, AKDD-4 and AKDD-6 showed the production of 6-APA from penicillin G. Assessment of production of 6-APA after incubation in penicillin G (2 mg/ml) by three different samples separately in free and agar immobilization state was done by HPLC analysis. Reusability of immobilized cells was found successful up to 14 days.