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Clavulanic acid is a medicinally important inhibitor of serine β-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC-MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Clavulanic acid (CA) is the preferred clinical drug for the treatment of infections by β-lactam antibiotic-resistant bacteria. CA is produced by Streptomyces clavuligerus, and although there have been many reports on the effects of carbon and nitrogen sources on CA production, the mechanisms involved remain unclear. In this study, we found that CA accumulation in S. clavuligerus F613-1 was increased significantly in MH medium, which is rich in organic nitrogen, compared with that in ML medium, which contains half the amount of organic nitrogen present in MH medium. Transcriptome analysis revealed that genes involved in CA biosynthesis, such as ceas1 , ceas2 , bls1 , bls2 , cas2 , pah2 , gcaS , and cad , and arginine biosynthesis, such as argB , argC , argD , argG , argH , argJ , and argR , were upregulated under rich organic nitrogen. Metabolome data revealed notable differences between cultures of F613-1 grown in MH and ML media with regard to levels of key intracellular metabolites, most of which are involved in arginine metabolic pathways, including arginine, glutamine, and glutamic acid. Additionally, supplementation of ML medium with arginine, glutamine, or glutamic acid resulted in increased CA production by S. clavuligerus F613-1. Our results indicate that rich organic nitrogen mainly affects CA biosynthesis by increasing the levels of amino acids associated with the arginine metabolic pathway and activating the expression of the CA biosynthetic gene cluster. These findings provide important insights for improving medium optimization and engineering of S. clavuligerus F613-1 for high-yield production of CA. IMPORTANCE The bacterium Streptomyces clavuligerus is used for the industrial production of the broad-spectrum β-lactamase inhibitor clavulanic acid (CA). However, much remains unknown about the factors which affect CA yields. We investigated the effects of different levels of organic nitrogen on CA production. Our analyses indicate that higher organic nitrogen levels were associated with increased CA yields and increased levels of arginine biosynthesis. Further analyses supported the relationship between arginine metabolism and CA production and demonstrated that increasing the levels of arginine or associated amino acids could boost CA yields. These findings suggest approaches for improving the production of this clinically important antibiotic.

The production of biocompounds through the cultivation of filamentous microorganisms is mainly affected by Oxygen Transfer Rate (OTR) and shear rate ([Formula: see text]) conditions. Despite efforts have been made to evaluate the effect of operating variables (impeller speed, N; and airflow rate, ϕ air ) on clavulanic acid production, no analysis regarding the effect of OTR and [Formula: see text] was made. Then, the aim of this study was to evaluate the dissociated effect of physical phenomena such as oxygen transfer and shear rate in the production of clavulanic acid from Streptomyces clavuligerus using a stirred tank bioreactor. Streptomyces clavuligerus cultivations were performed at five different OTR and [Formula: see text] conditions by manipulating the operating conditions (N, ϕ air , and gas inlet composition). Cultivations performed at equal impeller speed (600 rpm, similar [Formula: see text]) using oxygen enrichment, showed that CA productivity (Prod CA ) was positively affected by OTR increase. Subsequently, the different shear conditions (achieved by varying the impeller speed) lead to an increase in CA production levels. Despite both OTR and shear rate positively enhanced CA productivity, [Formula: see text] exhibited the highest impact: an increase of 145% in OTR initial enhanced the clavulanic acid productivity of about 29%, while an increment in the shear rate of 134% raised the Prod CA in 53%.

In this study, drug nanocarriers were designed using linear copolymers with different contents of cholinium-based ionic liquid units, i.e., [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TMAMA/Cl: 25, 50, and 75 mol%). The amphiphilicity of the copolymers was evaluated on the basis of their critical micelle concentration (CMC = 0.055-0.079 mg/mL), and their hydrophilicities were determined by water contact angles (WCA = 17°-46°). The chloride anions in the polymer chain were involved in ionic exchange reactions to introduce pharmaceutical anions, i.e., p -aminosalicylate (PAS - ), clavulanate (CLV - ), piperacillin (PIP - ), and fusidate (FUS - ), which are established antibacterial agents for treating lung and respiratory diseases. The exchange reaction efficiency decreased in the following order: CLV - > PAS - > PIP - >> FUS - . The hydrophilicity of the ionic drug conjugates was slightly reduced, as indicated by the increased WCA values. The major fraction of particles with sizes ~20 nm was detected in systems with at least 50% TMAMA carrying PAS or PIP. The influence of the drug character and carrier structure was also observed in the kinetic profiles of the release processes driven by the exchange with phosphate anions (0.5-6.4 μg/mL). The obtained polymer-drug ionic conjugates (especially that with PAS) are promising carriers with potential medical applications.

Background: Selective reactions to clavulanic acid (CLV) account for around 30% of immediate reactions after administration of amoxicillin-CLV. Currently, no immunoassay is available for detecting specific IgE to CLV, and its specific recognition in patients with immediate reactions has only been demonstrated by basophil activation testing, however with suboptimal sensitivity. The lack of knowledge regarding the structure of the drug that remains bound to proteins (antigenic determinant) is hampering the development of in vitro diagnostics. We aimed to identify the antigenic determinants of CLV as well as to evaluate their specific IgE recognition and potential role for diagnosis., Methods: Based on complex CLV degradation mechanisms, we hypothesized the formation of two antigenic determinants for CLV, AD-I (N-protein, 3-oxopropanamide) and AD-II (N-protein, 3-aminopropanamide), and designed different synthetic analogs to each one. IgE recognition of these structures was evaluated in basophils from patients with selective reactions to CLV and tolerant subjects. In parallel, the CLV fragments bound to proteins were identified by proteomic approaches., Results: Two synthetic analogs of AD-I were found to activate basophils from allergic patients. This determinant was also detected bound to lysines 195 and 475 of CLV-treated human serum albumin. One of these analogs was able to activate basophils in 59% of patients whereas CLV only in 41%. Combining both results led to an increase in basophil activation in 69% of patients, and only in 12% of controls., Conclusion: We have identified AD-I as one CLV antigenic determinant, which is the drug fragment that remains protein-bound., (© 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other β-lactamases are discussed.

The oppA2 gene encodes an oligopeptide-binding protein similar to the periplasmic substrate-binding proteins of the ABC transport systems. However, oppA2 is an orphan gene, not included in an ABC operon. This gene is located in the clavulanic acid (CA) gene cluster of Streptomyces clavuligerus and is essential for CA production. A transcriptomic study of the oppA2 -null mutant S. clavuligerus Δ oppA2 :: aac showed changes in the expression levels of 233 genes from those in the parental strain. These include genes for ABC transport systems, secreted proteins, peptidases, and proteases. Expression of the clavulanic acid, clavam, and cephamycin C biosynthesis gene clusters was not significantly affected in the oppA2 deletion mutant. The genes for holomycin biosynthesis were upregulated 2-fold on average, and the level of upregulation increased to 43-fold in a double mutant lacking oppA2 and the pSCL4 plasmid. Strains in which oppA2 was mutated secreted into the culture the compound N -acetylglycyl-clavaminic acid (AGCA), a putative intermediate of CA biosynthesis. A culture broth containing AGCA, or AGCA purified by liquid chromatography-mass spectrometry (LC-MS), was added to the cultures of various non-CA-producing mutants. Mutants blocked in the early steps of the pathway restored CA production, whereas mutants altered in late steps did not, establishing that AGCA is a late intermediate of the biosynthetic pathway, which is released from the cells when the oligopeptide-binding protein OppA2 is not available. IMPORTANCE The oppa2 gene encodes an oligopeptide permease essential for the production of clavulanic acid. A transcriptomic analysis of S. clavuligerus Δ oppA2 :: aac in comparison to the parental strain S. clavuligerus ATCC 27064 is reported. The lack of OppA2 results in different expression of 233 genes, including genes for proteases and genes for transport systems. The expression of the clavulanic acid genes in the oppA2 mutant is not significantly affected, but the genes for holomycin biosynthesis are strongly upregulated, in agreement with the higher holomycin production by this strain. The oppA2 -mutant is known to release N -acetylglycyl-clavaminic acid to the broth. Cosynthesis assays using non-clavulanic acid-producing mutants showed that the addition of pure N -acetylglycyl-clavaminic acid to mutants in which clavulanic acid formation was blocked resulted in the recovery of clavulanic acid production, but only in mutants blocked in the early steps of the pathway. This suggests that N -acetylglycyl-clavaminic acid is a previously unknown late intermediate of the clavulanic acid pathway., (Copyright © 2018 American Society for Microbiology.)

Clavulanate potassium (CP) is a classic β-lactamase inhibitor, which is extensively used as a combination drug in antibiotics therapy. As a chiral drug, CP is the only effective stereoisomer revealing prominent activities. To determine the chirality of CP efficiently, we provide an accurate and convenient method based on the comprehensive investigation of circular dichroism spectra and theoretical calculations. The results demonstrate two distinguishable cotton effects in electronic circular dichroism (ECD) and a sequence of cotton effect signs at specific wavenumbers in vibrational circular dichroism (VCD) can be regarded as diagnostic characteristics for the determination of absolute configuration. Additionally, the corresponding spectroscopic appearance of CP was elucidated with the help of theoretical analysis of molecular orbitals, natural bond orbitals and molecular electrostatic potential. The explicit absolute configuration determination of CP facilitates the thorough understanding in the stereochemistry of clavulanate potassium and its derivatives., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Clavulanate is used as an effective drug in combination with β-lactam antibiotics to treat infections of some antibiotic resistant bacteria. Here, we perform combined quantum mechanics/molecular mechanics simulations of several covalent complexes of clavulanate with class A β-lactamases KPC-2 and TEM-1. Simulations of the deacylation reactions identify the decarboxylated trans-enamine complex as being responsible for inhibition. Further, the obtained free energy barriers discriminate clinically relevant inhibition (TEM-1) from less effective inhibition (KPC-2).

In this work, the effects of agitation and aeration rates on aqueous two-phase system (ATPS)-based extractive fermentation of clavulanic acid (CA) by Streptomyces variabilis DAUFPE 3060 were investigated through a 2 2 full factorial design, where oxygen transfer rate (OTR) and oxygen uptake rate (OUR) were selected as the responses. Aeration rates significantly influenced cell growth, OUR, and CA yield, while OTR was practically the same in all the runs. Under the intermediate agitation (950 rpm) and aeration conditions (3.5 vvm) of the central point runs, it was achieved OTR of 1.617 ± 0.049 mmol L -1  h -1 , OUR of 0.132 ± 0.030 mmol L -1  h -1 , maximum CA production of 434 ± 4 mg L -1 , oxygen mass transfer coefficient of 33.40 ± 2.01 s -1 , partition coefficient of 66.5 ± 1.5, CA yield in the top and bottom phases of 75% ± 2% and 19% ± 1%, respectively, mass balance of 95% ± 4% and purification factor of 3.8 ± 0.1. These results not only confirmed the paramount role of O 2 supply, broth composition and operational conditions in CA ATPS-extractive fermentation, but also demonstrated the possibility of effectively using this technology as a cheap tool to simultaneously produce and recover CA. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1444-1452, 2016., (© 2016 American Institute of Chemical Engineers.)

One of the core goals of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) is to monitor major meat commodities for antimicrobial resistance. Targeted studies with methodologies based on core surveillance protocols are used to examine other foods, e.g., seafood, for antimicrobial resistance to detect resistances of concern to public health. Here we report the discovery of a novel Ambler class A carbapenemase that was identified in a nontoxigenic strain of Vibrio cholerae (N14-02106) isolated from shrimp that was sold for human consumption in Canada. V. cholerae N14-02106 was resistant to penicillins, carbapenems, and monobactam antibiotics; however, PCR did not detect common β-lactamases. Bioinformatic analysis of the whole-genome sequence of V. cholerae N14-02106 revealed on the large chromosome a novel carbapenemase (referred to here as VCC-1, for Vibrio cholerae carbapenemase 1) with sequence similarity to class A enzymes. Two copies of blaVCC-1 separated and flanked by ISVch9 (i.e., 3 copies of ISVch9) were found in an acquired 8.5-kb region inserted into a VrgG family protein gene. Cloned blaVCC-1 conferred a β-lactam resistance profile similar to that in V. cholerae N14-02106 when it was transformed into a susceptible laboratory strain of Escherichia coli. Purified VCC-1 was found to hydrolyze penicillins, 1st-generation cephalosporins, aztreonam, and carbapenems, whereas 2nd- and 3rd-generation cephalosporins were poor substrates. Using nitrocefin as a reporter substrate, VCC-1 was moderately inhibited by clavulanic acid and tazobactam but not EDTA. In this report, we present the discovery of a novel class A carbapenemase from the food supply., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

The kinetic and thermodynamic parameters of degradation of clavulanate potassium in the solid state were studied by using a reversed phase high performance liquid chromatography (RP-HPLC) method. The degradation of clavulanate potassium was a first-order reaction depending on the substrate concentration at an increased relative air humidity (RH) and in dry air. The dependence ln k = f(1/T) became the ln k = (0.026 ± 166.35)-(2702.82 ± 1779.43)(1/T) in dry air and ln k = (1.65 ± 100.40) × 10(3)-(5748.81 ± 3659.67)(1/T) at 76.4% RH. The thermodynamic parameters Ea, ΔH(≠a), ΔS(≠a) of the degradation of clavulanate potassium in the solid state were calculated. The dependence ln k = f (RH%) assumed the form ln k = (8.78 ± 5.75) 10 (-2) (RH%) + (2.64 × 10(-8 )± 40.41). The compatibility of clavulanate potassium with commonly used excipients was studied at an increased temperature and in dry air. The geometric structure of molecule, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals were also determined in order to predict the structural changes and reactive sites in clavulanate potassium during degradation and compatibility studies in the solid state. The ultraviolet (UV), Fourier transform infrared spectroscopy (FT-IR) and Raman spectra of degraded samples of the compound were analyzed.

Meropenem (MEM) and clavulanate potassium have been reported to demonstrate highly effective activity against Mycobacterium tuberculosis. There have been no reports on research into the complex of these chemotherapeutics concerning their mutually dependent stability or microbiological action on other microorganisms. Stability and compatibility studies of MEM/clavulanate potassium were conducted by using an HPLC-DAD method. The antibacterial activity of MEM/clavulanate potassium was tested in vitro against a selection of indicator bacteria strains by determining the MIC as well as analyzing the kinetics of changes in the concentrations of Pseudomonas aeruginosa, Staphylococcus aureus and Listeria monocytogenes caused by the action of MEM/clavulanate potassium. The stability and compatibility of MEM/clavulanate potassium were examined in aqua pro iniectione, 0.9% NaCl and 5% glucose at room temperature and at 5 °C. The degradation rates of MEM/clavulanate potassium depended on the type of infusion solvent used. Although in aqueous solutions of MEM/clavulanate potassium neither compound showed any mutual impact on the rate of degradation, clavulanate potassium was more labile than MEM. The synergy between these two resulted in a significantly lower value of MIC as compared to the values observed for the individual activity of either compound. The infusion solvent in which compatibility is observed between the components of the mixture MEM/clavulanate potassium is aqua pro iniectione. The complex MEM/clavulanate potassium demonstrates synergic antibacterial activity against P. aeruginosa, S. aureus and L. monocytogenes.

A stability-indicating LC assay method was developed and validated for a simultaneous determination of meropenem and potassium clavulanate in the presence of degradation products formed during acid-base hydrolysis, oxidation and thermolysis. The isocratic RP-HPLC method was developed with a LiChrospher RP-18 (250 mm x 4.6 mm, 5 microm) column and gradient elution of 12 mmol/L ammonium acetate and acetonitrile. The flow rate of the mobile phase was 1.0 mL/min, the detection wavelength 220 nm and the temperature 303 K. The method was validated with regard to linearity, accuracy, precision, selectivity and robustness, and was applied successfully for the determination of meropenem and potassium clavulanate separately as well as jointly in pharmaceutical formulations.

Growth of Microcystis aeruginosa could be inhibited significantly within 24 h by the extracellular substances prepared from Aeromonas sp. strain FM. During the treatment, the concentration of extracellular soluble carbohydrates increased significantly in algal culture. Morphological and ultrastructural changes in M. aeruginosa cells, including breakage of the cell surface, secretion of mucilage, and intracellular disorganization of thylakoids, were observed. HPLC-MS analysis showed that the extracellular substances of Aeromonas sp. strain FM were a mixture of free amino acids, tripeptides, and clavulanate. Among these, the algae-lysis effects of lysine and clavulanate were confirmed.

Structural and biochemical studies of the orf12 gene product (ORF12) from the clavulanic acid (CA) biosynthesis gene cluster are described. Sequence and crystallographic analyses reveal two domains: a C-terminal penicillin-binding protein (PBP)/β-lactamase-type fold with highest structural similarity to the class A β-lactamases fused to an N-terminal domain with a fold similar to steroid isomerases and polyketide cyclases. The C-terminal domain of ORF12 did not show β-lactamase or PBP activity for the substrates tested, but did show low-level esterase activity towards 3'-O-acetyl cephalosporins and a thioester substrate. Mutagenesis studies imply that Ser173, which is present in a conserved SXXK motif, acts as a nucleophile in catalysis, consistent with studies of related esterases, β-lactamases and D-Ala carboxypeptidases. Structures of wild-type ORF12 and of catalytic residue variants were obtained in complex with and in the absence of clavulanic acid. The role of ORF12 in clavulanic acid biosynthesis is unknown, but it may be involved in the epimerization of (3S,5S)-clavaminic acid to (3R,5R)-clavulanic acid.

New oral granules of amoxicillin and clavulanic acid in 8:1 ratio have recently been developed and approved to conduct clinical trial in China. To date, there has been no report studying the pharmacokinetic characteristics of amoxicillin and clavulanic acid in man. Therefore, it is urgent to investigate the pharmacokinetic properties of amoxicillin and clavulanic acid in man. The aim of the study was to assess the pharmacokinetic properties of amoxicillin and clavulanic acid in 8:1 with different dosage in healthy volunteers and provide support for this drug to obtain marketing authorization in China. A liquid chromatography-tandem mass spectrometry method for determining the concentration of amoxicillin and clavulanic acid in human plasma was developed and applied to this open-label, single- and multiple-dose Pharmacokinetics study. Subjects were randomized to receive a single dose of 1, 2, and 4 pouches of the test granulation of amoxicillin and clavulanic acid in 8:1 ratio (amoxicillin is 250 mg and clavulanic acid is 31.25 mg per pouch). In the single-dose phase, blood samples were collected before dosing and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 5, 8, 12, and 24 h after drug administration. In the multiple-dose phase, samples were obtained before drug administration on days 1, 2, 3, and 4 to determine the Cmin of amoxicillin and clavulanic acid. In the 4th day, samples were collected from 0.25 to 24 h after drug administration. Profiles of the concentration-time curves of amoxicillin and clavulanic acid were best fitted to two-compartment model. In this group of healthy Chinese subjects, the pharmacokinetics of amoxicillin fitted the linear dynamic feature at doses of 250,500 and 1,000 mg, and not obviously about clavulanic acid at doses of 31.25, 62.5, and 125 mg. The t 1/2 of single dose and multidoses were (1.45 ± 0.12) and (1.44 ± 0.26) h of amoxicillin and (1.24 ± 0.23) and (1.24 ± 0.17) of clavulanic acid, respectively; The AUC0-24 of single dose and multidoses were (27937.85 ± 4265.59) and (24569.80 ± 3663.63) ng h mL(-1) of amoxicillin and (891.45 ± 194.30) and (679.61 ± 284.05) ng h mL(-1) of clavulanic acid, respectively; The Cmax of single dose and multidoses were (8414.58 ± 1416.78) and (7929.17 ± 1291.54) ng mL(-1) of amoxicillin and (349.00 ± 89.54) and (289.00 ± 67.36) ng h mL(-1) of clavulanic acid, respectively. t 1/2, AUC0-24, and Cmax were similar after multiple-dose administration and after single-dose administration, suggesting that amoxicillin and clavulanic acid do not accumulate with multiple-dose administration of 500 and 62.5 mg, respectively.

The β-lactam antibiotics and related β-lactamase inhibitors are amongst the most important small molecules in clinical use. Most, but not all, β-lactams including penicillins, cephalosporins, and clavulanic acid are produced via fermentation or via modification of fermented intermediates, with important exceptions being the carbapenems and aztreonam. The desire for more efficient routes to existing antibiotics and for access to new and synthetically challenging ones stimulates continued interest in β-lactam biosynthesis. We review knowledge of the pathways leading to β-lactam antibiotics focusing on the mechanisms, structures and biocatalytic applications of the enzymes involved.

Objectives: Our aim was to unravel the inactivation pathway of the class A β-lactamase produced by Bacillus licheniformis BS3 (BS3) by clavulanate., Methods: The interaction between clavulanate and BS3 was studied by X-ray crystallography, pre-steady-state kinetics and mass spectrometry., Results: The analysis of the X-ray structure of the complex yielded by the reaction between clavulanate and BS3 indicates that the transient inactivated form, namely the cis-trans enamine complex, is hydrolysed to an ethane-imine ester covalently linked to the active site serine and a pentan-3-one-5-ol acid. It is the first time that this mechanism has been observed in an inactivated β-lactamase. Furthermore, the ionic interactions made by the carboxylic group of pentan-3-one-5-ol may provide an understanding of the decarboxylation process of the trans-enamine observed in the non-productive complex observed for the interaction between clavulanate and SHV-1 and Mycobacterium tuberculosis β-lactamase (Mtu)., Conclusions: This work provides a comprehensive clavulanate hydrolysis pathway accounting for the observed acyl-enzyme structures of class A β-lactamase/clavulanate adducts.

β-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of β-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A β-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with β-lactams and β-lactamase inhibitors. To better understand the contribution of these residues to the catalytic process, we explored the roles of R220 and E276 in KPC-2, a class A β-lactamase that inactivates carbapenems and β-lactamase inhibitors. Our study reveals that substitutions at R220 of KPC-2 selectively impact catalytic activity toward substrates (50% or greater reduction in k(cat)/K(m)). In addition, we find that residue 220 is central to the mechanism of β-lactamase inhibition/inactivation. Among the variants tested at Ambler position 220, the R220K enzyme is relatively "inhibitor susceptible" (K(i) of 14 ± 1 μM for clavulanic acid versus K(i) of 25 ± 2 μM for KPC-2). Specifically, the R220K enzyme is impaired in its ability to hydrolyze clavulanic acid compared to KPC-2. In contrast, the R220M substitution enzyme demonstrates increased K(m) values for β-lactamase inhibitors (>100 μM for clavulanic acid versus 25 ± 3 μM for the wild type [WT]), which results in inhibitor resistance. Unlike other class A β-lactamases (i.e., SHV-1 and TEM-1), the amino acid present at residue 276 plays a structural rather than kinetic role with substrates or inhibitors. To rationalize these findings, we constructed molecular models of clavulanic acid docked into the active sites of KPC-2 and the "relatively" clavulanic acid-susceptible R220K variant. These models suggest that a major 3.5-Å shift occurs of residue E276 in the R220K variant toward the active S70 site. We anticipate that this shift alters the shape of the active site and the positions of two key water molecules. Modeling also suggests that residue 276 may assist with the positioning of the substrate and inhibitor in the active site. These biochemical and molecular modeling insights bring us one step closer to understanding important structure-activity relationships that define the catalytic and inhibitor-resistant profile of KPC-2 and can assist the design of novel compounds.

The clavulanic acid is a substance which inhibits the β-lactamases used with penicillins for therapeutic treatment. After the fermentation, by-products of low molecular weight such as amino acids lysine, histidine, proline and tyrosine are present in the fermented broth. To remove these impurities the techniques of extraction by an aqueous two-phase system of 17% polyethylene glycol molecular weight 600 and 15% potassium phosphate were used for a partial purification. A subsequent ion-exchange adsorption was used for the recuperation of the clavulanic acid of the top phase and purification getting a concentration factor of 2 and purification of 100% in relation to the amino acids lysine, histidine, proline and tyrosine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Boronic acid transition state inhibitors (BATSIs) are potent class A and C β-lactamase inactivators and are of particular interest due to their reversible nature mimicking the transition state. Here, we present structural and kinetic data describing the inhibition of the SHV-1 β-lactamase, a clinically important enzyme found in Klebsiella pneumoniae, by BATSI compounds possessing the R1 side chains of ceftazidime and cefoperazone and designed variants of the latter, compounds 1 and 2. The ceftazidime and cefoperazone BATSI compounds inhibit the SHV-1 β-lactamase with micromolar affinity that is considerably weaker than their inhibition of other β-lactamases. The solved crystal structures of these two BATSIs in complex with SHV-1 reveal a possible reason for SHV-1's relative resistance to inhibition, as the BATSIs adopt a deacylation transition state conformation compared to the usual acylation transition state conformation when complexed to other β-lactamases. Active-site comparison suggests that these conformational differences might be attributed to a subtle shift of residue A237 in SHV-1. The ceftazidime BATSI structure revealed that the carboxyl-dimethyl moiety is positioned in SHV-1's carboxyl binding pocket. In contrast, the cefoperazone BATSI has its R1 group pointing away from the active site such that its phenol moiety moves residue Y105 from the active site via end-on stacking interactions. To work toward improving the affinity of the cefoperazone BATSI, we synthesized two variants in which either one or two extra carbons were added to the phenol linker. Both variants yielded improved affinity against SHV-1, possibly as a consequence of releasing the strain of its interaction with the unusual Y105 conformation.

(3R,5R)-Clavulanic acid (CA) is a clinically important inhibitor of Class A beta-lactamases. Sequence comparisons suggest that orf14 of the clavulanic acid biosynthesis gene cluster encodes for an acetyl transferase (CBG). Crystallographic studies reveal CBG to be a member of the emerging structural subfamily of tandem Gcn5-related acetyl transferase (GNAT) proteins. Two crystal forms (C2 and P2(1) space groups) of CBG were obtained; in both forms one molecule of acetyl-CoA (AcCoA) was bound to the N-terminal GNAT domain, with the C-terminal domain being unoccupied by a ligand. Mass spectrometric analyzes on CBG demonstrate that, in addition to one strongly bound AcCoA molecule, a second acyl-CoA molecule can bind to CBG. Succinyl-CoA and myristoyl-CoA displayed the strongest binding to the "second" CoA binding site, which is likely in the C-terminal GNAT domain. Analysis of the CBG structures, together with those of other tandem GNAT proteins, suggest that the AcCoA in the N-terminal GNAT domain plays a structural role whereas the C-terminal domain is more likely to be directly involved in acetyl transfer. The available crystallographic and mass spectrometric evidence suggests that binding of the second acyl-CoA occurs preferentially to monomeric rather than dimeric CBG. The N-terminal AcCoA binding site and the proposed C-terminal acyl-CoA binding site of CBG are compared with acyl-CoA binding sites of other tandem and single domain GNAT proteins., (2009 Wiley-Liss, Inc.)

As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

In this study, nanofiltration with NF200 membrane was employed to remove KCl from ion-exchanged solutions containing potassium clavulanate. The pore radius of NF200 membrane was estimated to be around 0.39 nm. The effects of operating pressure on separation performance were investigated in a range of 100-400 psig. The influences of cross-flow velocity (0.14-0.70 cm/s), temperature (4-25 degrees C), and feed composition were also investigated. In all experiments, clavulanate rejection showed high levels from 0.91 to 0.99, while chloride rejection ranged from 0.06 to 0.54. In a case at an operating pressure of 50 psig and 25 degrees C, as much as 94% of clavulanate was retained while 94% of chloride was removed, indicating that NF200 membrane was a suitable choice for selectively removing KCl. NF200 membrane also showed a stable performance in the operational stability test with an ion-exchanged solution obtained by treating actual fermentation broth.

The Great White Plague: Mycobacterium tuberculosis, the bacteria causing tuberculosis, is a continuing threat to global health through the emergence of resistant strains and the lack of novel therapeutic agents. Recently reported results on this important work are highlighted.

The stability of aqueous admixtures of amoxicillin sodium and potassium clavulanate was studied in the liquid state at selected pH values. Potassium clavulanate was found to catalyze the rate of degradation of amoxicillin sodium under the conditions of this study. In phosphate buffer (at pH 7.0) both amoxicillin sodium and potassium clavulanate showed first-order degradation when stored separately. However, when combined the rate of amoxicillin degradation increased and t(90) values for amoxicillin decreased from 69.6 min for amoxicillin alone to 10.8 min for amoxicillin in the combination at 55 degrees C. A kinetic model was developed that explained the catalytic behavior of potassium clavulanate and phosphate buffer. In acetate buffer the rate of degradation of amoxicillin sodium followed first-order kinetics, but the catalytic effect of clavulanate caused curvature in the rate plots at higher temperatures and clavulanate concentrations. This catalytic effect was less than that occurred in phosphate buffer (where the t(90) value of amoxicillin decreased from 137.3 min for amoxicillin alone to 52.5 min for amoxicillin in combination at 55 degrees C). First-order bi-exponential decay occurred with amoxicillin degradation, which explained this change in rate.

Molecular dynamics (MD) simulation and quantum chemical (QC) calculations were used to investigate the reaction mechanism of the formation of acyclic clavulanate intermediates in the inhibition of class A beta-lactamase. The initial model for QC calculations was derived from an MD simulation. It was composed of a substrate clavulanate and four residues (Ser70, Gln237, Ser130, and Ser216), which form hydrogen bonds with the substrate. The QC calculation results indicate that the oxazolidine ring can undergo cleavage by proton transfer, which yields not only imine but also enamine products. A new mechanism involving hydrogen transfer from C6 to O1 has been suggested. Besides, MD simulation provided evidence that the water molecule can catalyze the proton transfer, and QC calculation shows water assistance can decrease the energy barrier greatly.

Tazobactam, sulbactam, and clavulanic acid are the only beta-lactamase inhibitors in clinical use. Comparative inhibitory activities of clavulanic acid, sulbactam, and tazobactam against clinically important beta-lactamases conclude that tazobactam is superior to both clavulanic acid and sulbactam. Thus far, the majority of explanations for this phenomenon have relied on kinetic studies, which report differences in the ligands' apparent dissociation constants and number of turnovers before inactivation. Due their innate limitations, these investigations do not examine the identity of intermediates on the reaction pathway and relate them to the efficacy of the inhibitors. In the present study, the reactions between the three inhibitors and SHV-1 beta-lactamase have been examined in single crystals using a Raman microscope. The results show that tazobactam forms a predominant population of trans-enamine, a chemically inert species, with SHV-1, while clavulanate and sulbactam form a mixture of trans-enamine and two labile species, the cis-enamine and imine. The same reactions are then reexamined using a deacylation-deficient variant, SHV E166A, that has been used to trap acyl-enzyme intermediates for X-ray crystallographic analysis. Our Raman data show that significant differences exist between the wild-type and SHV E166A acyl-enzyme populations. Namely, compared to SHV-1, sulbactam shows significantly smaller populations of cis-enamine and imine in the E166A variant, while clavulanate exists almost exclusively as trans-enamine in the E166A active site. Using clavulanate as an example, we also show that Raman crystallography can provide novel information on the presence of multiple conformers or tautomers for intermediates within a complex reaction pathway. These insights caution against the interpretation of experimental data obtained with deacylation-deficient beta-lactamases to make mechanistic conclusions about inhibitors within the enzyme.

Clavulanic acid is a widely used beta-lactamase inhibitor whose key beta-lactam core is formed by beta-lactam synthetase. beta-Lactam synthetase exhibits a Bi-Ter mechanism consisting of two chemical steps, acyl-adenylation followed by beta-lactam formation. 32PPi-ATP exchange assays showed the first irreversible step of catalysis is acyl-adenylation. From a small, normal solvent isotope effect (1.38 +/- 0.04), it was concluded that beta-lactam synthesis contributes at least partially to kcat. Site-specific mutation of Lys-443 identified this residue as the ionizable group at pKa approximately 8.1 apparent in the pH-kcat profile that stabilizes the beta-lactam-forming step. Viscosity studies demonstrated that a protein conformational change was also partially rate-limiting on kcat attenuating the observed solvent isotope effect on beta-lactam formation. Adherence to Kramers' theory gave a slope of 1.66 +/- 0.08 from a plot of log(o kcat/kcat) versus log(eta/eta(o)) consistent with opening of a structured loop visible in x-ray data preceding product release. Internal "friction" within the enzyme contributes to a slope of > 1 in this analysis. Correspondingly, earlier in the catalytic cycle ordering of a mobile active site loop upon substrate binding was manifested by an inverse solvent isotope effect (0.67 +/- 0.15) on kcat/Km. The increased second-order rate constant in heavy water was expected from ordering of this loop over the active site imposing torsional strain. Finally, an Eyring plot displayed a large enthalpic change accompanying loop movement (DeltaH approximately 20 kcal/mol) comparable to the chemical barrier of beta-lactam formation.

Natural antibiotics are almost universal secondary metabolites, not essential for the growth of the producing organisms generally produced at low growth rates or after growth has ceased. Clavulanic acid (CA), a naturally occurring powerful inhibitor of bacterial beta-lactamases is a major beta-lactam antibiotic produced by organism Streptomyces clavuligerus and is active against a wide spectrum of Gram-positive and Gram-negative bacteria. The review discusses the biosynthetic pathway, fermentative production, downstream processing and applications of CA.

The intrinsic resistance of Mycobacterium tuberculosis to the beta-lactam class of antibiotics arises from a chromosomally encoded, extended spectrum, class A beta-lactamase, BlaC. Herein, we report the X-ray crystallographic structure of BlaC inhibited with clavulanate at a resolution of 1.7 A with an R-factor value of 0.180 and R-free value of 0.212 for the m/ z +154 clavulanate-derived fragment observed in the active site. Structural evidence reveals the presence of hydrogen bonds to the C1 carbonyl along with a coplanar arrangement of C1, C2, C3, and N4, which favors enolization to generate a trans-alpha,beta-eneamine, stabilizing the +154 adduct from hydrolysis. The irreversible inhibition of BlaC suggests that treatment of M. tuberculosis with a combination of a beta-lactam antibiotic and clavulanate may lead to rapid bactericidal activity.

Amino acid changes at Ambler position R244 in class A TEM and SHV beta-lactamases confer resistance to ampicillin/clavulanate, a beta-lactam/beta-lactamase inhibitor combination used to treat serious infections. To gain a deeper understanding of this resistance phenotype, we investigated the activities of sulbactam and two novel penem beta-lactamase inhibitors with sp2 hybridized C3 carboxylates and bicyclic R1 side chains against a library of SHV beta-lactamase variants at the 244 position. Compared to SHV-1 expressed in Escherichia coli, all 19 R244 variants exhibited increased susceptibility to ampicillin/sulbactam, an important difference compared to ampicillin/clavulanate. Kinetic analyses of SHV-1 and three SHV R244 (-S, -Q, and -L) variants revealed the Ki for sulbactam was significantly elevated for the R244 variants, but the partition ratios, kcat/kinact, were markedly reduced (13 000 -->

Kinetics of the reactions of amoxicillin sodium and potassium clavulanate alone and in combination were investigated in the frozen state at selected pH values of 2.0, 4.6 and 7.0. Extrapolation of the rate constant values to the frozen state from the liquid state data indicated marked acceleration of the rates of amoxicillin and clavulanate degradation for the pH values investigated. The highest acceleration in rate recorded was 15.0-fold for clavulanate and the lowest value was 4.6-fold for amoxicillin at -7.3 degrees C in the hydrochloric acid system. The rate constant values obtained were interpreted in terms of the concentration model [Pincock, R.E., Kiovsky, T.E., 1966. Kinetics of reactions in frozen solution. J. Chem. Educ. 43, 358-360], phase-temperature relationship of the solutes, buffer catalysis, pH change and polymerization reactions. A kinetic model was deduced for the hydrochloric acid system providing adequate explanation of the experimental results. A large stabilizing effect of sodium chloride used for maintaining constant ionic strength (micro=0.5) was evident in this system. The shelf-life of amoxicillin was increased from 2.2 to 58.7h at -7.3 degrees C when sodium chloride was included in the hydrochloric acid system.

Sulbactam is a mechanism-based inhibitor of beta-lactamase enzymes used in clinical practice. It undergoes a complex series of chemical reactions in the active site that have been studied extensively in the past three decades. However, the actual species that gives rise to inhibition in a clinical setting has not been established. Recent studies by our group, using Raman microscopy and X-ray crystallography, have found that large quantities of enamine-based acyl-enzyme species are present within minutes in single crystals of SHV-1 beta-lactamases which can lead to significant inhibition. The enamines are formed by breakdown of the cyclic beta-lactam structures with further transformations leading to imine formation and subsequent isomerization to cis and/or trans enamines. Another favored form of inhibition arises from attack on the imine by a second nucleophilic amino acid side chain, e.g., from serine 130, to form a cross-linked species in the active site that can degrade to an acrylate-like species irreversibly bound to the enzyme. Thus, the imine is at a branch point on the reaction pathway. Using sulbactam and 6,6-dideuterated sulbactam we follow these alternate paths in WT and E166A SHV-1 beta-lactamase by means of Raman microscopic studies on single enzyme crystals. For the unlabeled sulbactam, the Raman data show the presence of an acrylate-like species, probably 3-serine acrylate, several hours after the reaction is started in the crystal. However, for the 6,6-dideutero analogue the acrylate signature appears on the time scale of minutes. The Raman signatures, principally an intense feature near 1530 cm-1, are assigned based on quantum mechanical calculations on model compounds that mimic acrylate species in the active site. The different time scales observed for acrylate-like product formation are ascribed to different rates of reaction involving the imine intermediate. It is proposed that for the unsubstituted sulbactam the conversion from imine to enamine, which involves breaking a C-H bond, is aided by quantum mechanical tunneling. For the 6,6-dideutero-sulbactam the same step involves breaking a C-D bond, which has little or no assistance from tunneling. Consequently the conversion to enamines is slower, and a higher population of imine results, presenting the opportunity for the competing reaction with the second nucleophile, serine 130 being the prime candidate. The hydrolysis of the resulting cross-linked intermediate leads to the observed rapid buildup of the acrylate product in the Raman spectra from the dideutero analogue. The protocol used here, essentially running the reactions with the two forms of sulbactam in parallel, provides an element of control and enables us to conclude that, for the unsubstituted sulbactam, the formation of the cross-linked intermediate and the final irreversible acrylate product is not a significant route to inhibition of SHV-1.

The purpose of this study was to develop and validate a method for the determination of clavulanic acid (CLAV) residues in edible tissues of swine by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS). After a simple extraction of CLAV using an aqueous phosphate buffer solution of pH 6.0, an ultrafiltration step was performed for protein removal. Chromatography of CLAV and the internal standard tazobactam (TAZO) was achieved on a reversed-phase PLRP-S polymeric column (150 mm x 2.1 mm i.d., 100 A) using a mixture of 0.05 (v/v)% formic acid in water and acetonitrile. The mass spectrometer was operated in the MS/MS selected reaction monitoring (SRM) mode. The method was validated for the analysis of porcine muscle, skin plus fat, liver and kidney, according to the requirements defined by the European Community. Calibration curves were prepared for all tissues and good linearity was achieved over the concentration ranges tested (correlation coefficient > or = 0.99 and goodness-of-fit coefficient < or = 10%). Limits of quantification of 50 ng g(-1) were obtained for the analysis of CLAV in the various tissues which corresponds in all cases to at least half the maximum residue limits (MRLs). Limits of detection ranged between 8.0 and 15.14 ng g(-1). The within-day, between-day precisions and trueness fell within the ranges specified in the EMEA/CVMP/573-00/FINAL document. Biological samples from pigs that received an oral or intravenous bolus of a commercial amoxicillin/clavulanic acid formulation were analyzed using the described method.

Antibiotic resistance to beta-lactam compounds in Gram-negative bacteria such as Escherichia coli and Klebsiella pneumoniae is often mediated by beta-lactamase enzymes like TEM and SHV. Previously, a limited number of inhibitors have shown efficacy in combating such bacterial drug resistance. However, many Gram-negative pathogens have evolved inhibitor resistant forms of these hydrolytic enzymes. A single point mutation of the active site residue Ser130 to a Gly in either TEM or SHV results in resistance to amoxicillin and clavulanic acid, an important clinical beta-lactam-beta-lactamase inhibitor combination antibiotic. Previous structural and modeling studies of the S130G mutants of TEM and SHV have shown differences in how these two distinct but closely related enzymes compensate for the loss of the Ser130 residue. In the case of S130G SHV, a structure of tazobactam in the active site has suggested that the inhibitor preferentially assumes a cis-enamine intermediate form when the Ser130 hydroxyl is absent. Raman crystallographic studies of S130G SHV inhibited with tazobactam, sulbactam, clavulanic acid, and 2'-glutaroxy penem sulfone (SA2-13) were performed with the aim of identifying the type and amount of intermediate formed with each drug to understand the role of the S130G mutation in formation of the important enamine intermediates. It is demonstrated that with the exception of sulbactam, each compound forms observable trans-enamine intermediates. For S130G reacted with tazobactam, identical steady state levels of enamine are achieved when compared to those of wild-type (WT) or even deacylation deficient forms of the enzyme. With clavulanic acid, slightly smaller amounts of enamine are observed within the first 30 min of the reaction but are not significantly different than those for tazobactam. Thus, the resistance mutation does not substantially affect the amount of trans-enamine formed with clavulanic acid during the critical early time period of inhibition. This finding has important implications in the design of beta-lactamase inhibitors for drug resistant variants like S130G SHV.

The present work reports the nutritional requirements and environmental conditions for submerged culture of Streptomyces clavuligerus for clavulanic acid production using orthogonal matrix method (Taguchi L(16) design) and also fed-batch fermentation for clavulanic acid production by feeding glycerol, arginine and threonoine to the fermentation medium intermittently. Clavulanic acid production was increased by 18% with the span of feeding glycerol and reached a maximum at 1.30mg/ml with 120h glycerol feeding as compared to 1.10mg/ml in the control. The production also increased with the span of feeding amino acids and reached a maximum of 1.31 and 1.86mg/ml with feeding arginine and threonine, respectively in 120h. There was an overall increase of 18% and 9% in clavulanic acid production with arginine and threonine feeding as compared to the respective controls (1.10 and 1.70mg/ml, respectively).

The mechanism of the oxidative cyclization reaction catalyzed by clavaminic acid synthase (CAS) was studied in silico. First, a classical molecular dynamics (MD) simulation was performed to obtain a realistic structure of the CAS-Fe(IV)=O-succinate-substrate complex; then potential of mean force (PMF) was calculated to assess the feasibility of the beta-lactam ring, more specifically its C4' corner, approaching the oxo atom. Based on the MD structure, a relatively large model of the active site region was selected and used in the B3LYP investigation of the reaction mechanism. The computational results suggest that once the oxoferryl species is formed, the oxidative cyclization catalyzed by CAS most likely involves either a mechanism involving C4'(S)-H bond cleavage of the monocyclic beta-lactam ring, or a biosynthetically unprecedented mechanism comprising (1) oxidation of the hydroxyl group of PCA to an O-radical, (2) retro-aldol-like decomposition of the O-radical to an aldehyde and a C-centered radical, which is stabilized by the captodative effect, (3) abstraction of a hydrogen atom from the C4'(S) position of the C-centered radical by the Fe(III)-OH species yielding an azomethine ylide, and (4) 1,3-dipolar cycloaddition to the ylide with aldehyde acting as a dipolarophile. Precedent for the new proposed mechanism comes from the reported synthesis of oxapenams via 1,3-dipolar cycloaddition reactions of aldehydes and ketones.

The calculation of the vibrational Raman spectrum of enzyme-bound beta-lactamase inhibitors may be of help to understand the mechanisms responsible for bacterial drug resistance. Here, we present a study of the solvation structure and the vibrational properties of clavulanate, an important beta-lactamase inhibitor, in aqueous solution as obtained from full quantum and hybrid empirical/quantum molecular dynamics simulations at ambient conditions. The analysis of the vibrational density of states indicates that hybrid empirical/quantum mechanical simulations are able to properly describe the vibrational levels of clavulanate in solution. In addition, we propose a computationally efficient protocol to calculate the vibrational Raman effect for large solute molecules in water, which is able to faithfully reproduce the experimentally recorded clavulanate Raman spectrum and discloses the possibility to employ hybrid simulations to assign the experimental Raman spectra of inhibitors bound to beta-lactamases.

Clavulanic acid is a naturally occurring antibiotic produced by Streptomyces clavuligerus. The present work reports on clavulanic acid production by Streptomyces clavuligerus MTCC 1142 using one-factor-at-a-time and L(25) orthogonal array. The one-factor-at-a-time method was adopted to investigate the effect of media components (i.e., carbon source, nitrogen source and inoculum concentration) and environmental factors such as pH for clavulanic acid production. Production of clavulanic acid by Streptomyces clavuligerus was investigated using seven different carbon sources (viz. glucose, sucrose, modified starch, rice-bran oil, soybean oil, palm oil, and glycerol) and six different nitrogen sources (viz. peptone, yeast extract, ammonium chloride, ammonium carbonate, sodium nitrate and potassium nitrate). A maximum yield of 140 microg/mL clavulanic acid was obtained in the medium containing soybean oil as a carbon source and yeast extract as nitrogen source. Subsequently, the concentration of soybean flour, soybean oil, dextrin, yeast extract and K2HPO4 were optimized using L25 orthogonal array method. The final optimized medium produced 500 microg/mL clavulanic acid at the end of 96 h as compared to 140 microg/mL before optimization. Synthesis of precursor molecules as a metabolic driving force is of considerable importance in antibiotic synthesis. Attempts to increase the clavulanic acid synthesis by manipulating the anaplerotic flux on C(3) and C(5) precursors by supplementing the medium with arginine, ornithine, proline, valine, leucine, isoleucine, pyruvic acid and alpha-ketoglutarate were successful. Supplementing the optimized medium with 0.1 M arginine and 0.1 M leucine increased the yield of clavulanic acid further to 1100 microg/mL and 1384 microg/mL respectively.

(3R,5R)-Clavulanic acid, a clinically used inhibitor of serine beta-lactamases, is produced by fermentation of Streptomyces clavuligerus. The early steps in clavulanic acid biosynthesis leading to the bicyclic beta-lactam intermediate (3S,5S)-clavaminic acid have been defined. However, the mechanism by which (3S,5S)-clavaminic acid is converted to the penultimate intermediate (3R,5R)-clavaldehyde is unclear. Disruption of orf15 or orf16, of the clavulanic acid biosynthesis gene cluster, blocks clavulanic acid production and leads to the accumulation of N-acetyl-glycyl-clavaminic acid and N-glycyl-clavaminic acid, suggesting that these compounds are intermediates in the pathway. Two alternative start codons have been proposed for orf17 to encode for two possible polypeptides, one of which has 92 N-terminal residues less then the other. The shorter version of orf17 was successfully expressed in Escherichia coli and purified as a monomeric protein. Sequence analyses predicting the ORF17 protein to be a member of the ATP-grasp fold superfamily were supported by soft ionization mass spectrometric analyses that demonstrated binding of ATP to the ORF17 protein. Semisynthetic clavaminic acid, prepared by in vitro reconstitution of the biosynthetic pathway from the synthetically accessible intermediate proclavaminic acid, was shown by mass spectrometric analyses to be converted to N-glycyl-clavaminic acid in the presence of ORF17, ATP, and glycine. Under the same conditions N-acetyl-glycine and clavaminic acid were not converted to N-acetyl-glycyl-clavaminic acid. The specificity of ORF17 as an N-glycyl-clavaminic acid synthetase, together with the reported accumulation of N-glycyl-clavaminic acid in orf15 and orf16 disruption mutants, suggested that N-glycyl-clavaminic acid is an intermediate in clavulanic acid biosynthesis.

The information content of organic molecules has been the subject of a series of papers from this lab. The investigation continues as aggregation states and some of their attendant solvent effects are examined. An organic molecule operates as an information source not in a vacuum but rather in conjunction with one or more solvent compounds. Accordingly, solvents (ethanol, acetone, etc.) furnish both a source and a channel that modify molecular information. In this paper, Brownian techniques are developed further so as to quantify molecular information with aggregation states taken into account. Several applications follow concerning organic acid ionization in solution, tautomerization reactions, and molecular activity at biological receptor sites. The goal is to advance Brownian processing as a means of probing molecular information and its communication. To this end, practical examples are offered relating structure and function along informatic lines.

Antibiotic formulation effluent is well known for its important contribution to environmental pollution due to its fluctuating and recalcitrant nature. In the present study, the chemical treatability of penicillin formulation effluent (average filtered COD(o)=830 mg/l; average soluble COD(o)=615 mg/l; pH(o)=6.9) bearing the active substances penicillin Amoxicillin Trihydrate (C(16)H(19)N(3)O(5)S.3H(2)O) and the beta-lactamase inhibitor Potassium Clavulanate (C(8)H(8)KNO(5)) has been investigated. For this purpose, the penicillin formulation effluent was subjected to ozonation (applied ozone dose=2500 mg/(lxh)) at varying pH (2.5-12.0) and O(3)+H(2)O(2) (perozonation) at different initial H(2)O(2) concentrations (=2-40 mM) and pH 10.5. According to the experimental results, the overall Chemical Oxygen Demand (COD) removal efficiency varied between 10 and 56% for ozonation and 30% (no H(2)O(2)) and 83% (20 mM H(2)O(2)) for the O(3)+H(2)O(2) process. The addition of H(2)O(2) improved the COD removal rates considerably even at the lowest studied H(2)O(2) concentration. An optimum H(2)O(2) concentration of 20 mM existed at which the highest COD removal efficiency and abatement kinetics were obtained. The ozone absorption rate ranged between 53% (ozonation) and 68% (perozonation). An ozone input of 800 mg/l in 20 min was sufficient to achieve the highest BOD(5)/COD (biodegradability) ratio (=0.45) and BOD(5) value (109 mg/l) for the pre-treated penicillin formulation effluent. After the establishment of optimum ozonation and perozonation conditions, mixtures of synthetic domestic wastewater+raw, ozonated and perozonated penicillin formulation effluent were subjected to biological activated sludge treatment at a food-to-microorganisms (F/M) ratio of 0.23 mg COD/(mg MLSSxd), using a consortium of acclimated microorganisms. COD removal efficiencies of the activated sludge process were 71, 81 and 72% for pharmaceutical wastewater containing synthetic domestic wastewater mixed with either raw, ozonated or perozonated formulation effluent, respectively. The ultimate COD value obtained after 24-h biotreatment of the synthetic domestic wastewater+pre-ozonated formulation effluent mixture was around 100 mg/l instead of 180 mg/l which was the final COD obtained for the wastewater mixture containing raw formulation effluent, indicating that pre-ozonation at least partially removed the non-biodegradable COD fraction of the formulation effluent.

The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution. The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids.

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.

The industrial production of beta-lactam antibiotics by fermentation over the past 50 years is one of the outstanding examples of biotechnology. Today, the beta-lactam antibiotics, particularly penicillins and cephalosporins, represent the world's major biotechnology products with worldwide dosage form sales of approximately 15 billion US dollars or approximately 65% of the total world market for antibiotics. Over the past five decades, major improvements in the productivity of the producer organisms, Penicillium chrysogenum and Acremonium chrysogenum (syn. Cephalosporium acremonium) and improved fermentation technology have culminated in enhanced productivity and substantial cost reduction. Major fermentation producers are now estimated to record harvest titers of 40-50 g/l for penicillin and 20-25 g/l for cephalosporin C. Recovery yields for penicillin G or penicillin V are now >90%. Chemical and enzymatic hydrolysis process technology for 6-aminopenicillanic acid or 7-aminocephalosporanic acid is also highly efficient (approximately 80-90%) with new enzyme technology leading to major cost reductions over the past decade. Europe remains the dominant manufacturing area for both penicillins and cephalosporins. However, due to ever increasing labor, energy and raw material costs, more bulk manufacturing is moving to the Far East, with China, Korea and India becoming major production countries with dosage form filling becoming more dominant in Puerto Rico and in Ireland.

A micellar electrokinetic chromatography method for simultaneous assay of ticarcillin and clavulanic acid in Timentin i.v. injection preparation was developed. This method ensures excellent separation of both components of Timentin preparation. The validation of the method was performed, and specificity, reproducibility, precision and accuracy were confirmed. The detection and quantitative limits for Timentin were established in the concentrations 0.04 and 0.08 mg/ml, respectively. The elaborated technique was compared with two methods routinely used-UV and high performance liquid chromatography (HPLC). The obtained results and their statistical analysis proved the same precision of all methods, however, no significant differences were observed between CE and HPLC.

The aims of this investigation were to calculate the pharmacokinetic parameters of amoxicillin and clavulanic acid, and to identify parameters that may affect their observed differences in absorption. Data were obtained from plasma concentration-time curves from four different open, randomized, two-treatment, two-period, two-sequence, crossover Phase I bioequivalence studies, with the following co-amoxiclav formulations: tablets 250/125, 500/125 and 875/125 mg, or 10 mL of an oral suspension 250/62.5 mg per 5 mL. Data from 144 subjects and 288 drug administrations were available for evaluation. After a 125 mg clavulanic acid dose (administered as potassium clavulanate) for all four different formulations, the clavulanic acid AUC(t) data ranged from 1.5 to 8 mg.h/L, varying by a factor of 5. The absorption of clavulanic acid was not related to the absorption of amoxicillin, or demographic factors, and we were unable to identify the reasons for the large variability in the absorption of clavulanic acid. We conclude that the absorption of clavulanic acid, after oral administration, is highly variable and may vary over a five-fold range between patients.

To help elucidate why penicillin-G is inactivated by certain bacterial beta-lactamase enzymes, whereas clavulanic acid (Clav, which is similar to penicillin-G except at positions 1, 2, and 6) inhibits beta-lactamase, the intrinsic chemical reactivities of these two antibiotics were assessed in this work. Ab initio and continuum dielectric methods were used to map out the gas-phase and solution-phase free-energy profiles for the alkaline hydrolyses of Clav and penicillanic acid (Peni, which is similar to penicillin-G except at position 6) as well as of a fictitious hybrid compound, Peni-db, which is similar to Clav and Peni except at positions 1 and 2, respectively. Furthermore, the ring strain energies of various lactam rings and the five-membered rings of Peni and Clav as well as their respective rate-limiting transition states were computed to assess the contribution of four- and five-membered ring strains to the antibiotic's activity. The predicted product distribution, rate-limiting step, and relative reaction rates for the alkaline hydrolysis of Peni and Clav are in accord with the experimental findings. The rate-limiting step in the alkaline hydrolysis of Peni, Clav, or Peni-db is the approach of the negatively charged hydroxide ion toward the anionic reactant to form a tetrahedral intermediate. The alkaline hydrolysis of Clav generates more stable products than that of Peni mainly because the O1 atom and the hydroxyethylidene group in Clav facilitate the opening of the five-membered ring; furthermore, the O1 atom can abstract a proton easier than the less polar S1 in Peni. Clav undergoes basic hydrolysis faster than Peni mainly because its hydroxyethylidene group leads to an increase in the positive charge on the carbonyl C7 atom, therefore enhancing favorable electrostatic interactions with the incoming hydroxide anion. To a lesser extent, the oxygen at position 1 in Clav also contributes to the rate acceleration because of the greater solvent stabilization of the oxygen-containing transition state as compared to the respective ground state. The inherent strain of the four-membered beta-lactam ring or five-membered ring does not enhance the alkaline hydrolyses of beta-lactam molecules such as Peni or Clav, consistent with the observation that the rate-limiting step does not involve a breakdown of the four-membered beta-lactam ring or five-membered thiazolidine/oxazolidine rings.