Rich Organic Nitrogen Impacts Clavulanic Acid Biosynthesis through the Arginine Metabolic Pathway in Streptomyces clavuligerus F613-1.

Clavulanic acid (CA) is the preferred clinical drug for the treatment of infections by ß-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 ß-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.

Individual effect of shear rate and oxygen transfer on clavulanic acid production by Streptomyces clavuligerus.

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, ϕair) 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, ϕair, and gas inlet composition). Cultivations performed at equal impeller speed (600 rpm, similar [Formula: see text]) using oxygen enrichment, showed that CA productivity (ProdCA) 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 OTRinitial enhanced the clavulanic acid productivity of about 29%, while an increment in the shear rate of 134% raised the ProdCA in 53%.

Linear Copolymers Based on Choline Ionic Liquid Carrying Anti-Tuberculosis Drugs: Influence of Anion Type on Physicochemical Properties and Drug Release.

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.

Identification of an antigenic determinant of clavulanic acid responsible for IgE-mediated reactions.

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.

New Conformations of Acylation Adducts of Inhibitors of ß-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to ß-lactam antibiotics due to the production of the extended spectrum ß-lactamase BlaC. ß-Lactam/ß-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 ß-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 ß-lactamases are discussed.

Transcriptional Studies on a Streptomyces clavuligerus oppA2 Deletion Mutant: N-Acetylglycyl-Clavaminic Acid Is an Intermediate of Clavulanic Acid Biosynthesis.

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 ΔoppA2::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 ΔoppA2::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.

Diagnostic absolute configuration determination of clavulanate potassium: A comprehensive investigation of chiroptical spectroscopies and theoretical calculations.

Clavulanate potassium (CP) is a classic ß-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.

Multiscale Simulations of Clavulanate Inhibition Identify the Reactive Complex in Class A ß-Lactamases and Predict the Efficiency of Inhibition.

Clavulanate is used as an effective drug in combination with ß-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 ß-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).

Effect of aeration and agitation on extractive fermentation of clavulanic acid by using aqueous two-phase system.

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 22 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 O2 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.

Characterization of VCC-1, a Novel Ambler Class A Carbapenemase from Vibrio cholerae Isolated from Imported Retail Shrimp Sold in Canada.

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 ß-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 ß-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.

Stability, compatibility and microbiological activity studies of meropenem-clavulanate potassium.

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.

Solid-state stability and compatibility studies of clavulanate potassium.

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.

Development and validation of stability-indicating HPLC method for simultaneous determination of meropenem and potassium clavulanate.

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.

Structural and mechanistic studies of the orf12 gene product from the clavulanic acid biosynthesis pathway.

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)/ß-lactamase-type fold with highest structural similarity to the class A ß-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 ß-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, ß-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.

Inhibition of Microcystis aeruginosa by the extracellular substances from an Aeromonas sp.

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.

Pharmacokinetics study of amoxycillin and clavulanic acid (8:1)--a new combination in healthy Chinese adult male volunteers using the LC-MS/MS method.

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 enzymes of ß-lactam biosynthesis.

The ß-lactam antibiotics and related ß-lactamase inhibitors are amongst the most important small molecules in clinical use. Most, but not all, ß-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 ß-lactam biosynthesis. We review knowledge of the pathways leading to ß-lactam antibiotics focusing on the mechanisms, structures and biocatalytic applications of the enzymes involved.

Novel fragments of clavulanate observed in the structure of the class A ß-lactamase from Bacillus licheniformis BS3.

OBJECTIVES: Our aim was to unravel the inactivation pathway of the class A ß-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 ß-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 ß-lactamase (Mtu). CONCLUSIONS: This work provides a comprehensive clavulanate hydrolysis pathway accounting for the observed acyl-enzyme structures of class A ß-lactamase/clavulanate adducts.

Understanding the molecular determinants of substrate and inhibitor specificities in the Carbapenemase KPC-2: exploring the roles of Arg220 and Glu276.

ß-Lactamases are important antibiotic resistance determinants expressed by bacteria. By studying the mechanistic properties of ß-lactamases, we can identify opportunities to circumvent resistance through the design of novel inhibitors. Comparative amino acid sequence analysis of class A ß-lactamases reveals that many enzymes possess a localized positively charged residue (e.g., R220, R244, or R276) that is critical for interactions with ß-lactams and ß-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 ß-lactamase that inactivates carbapenems and ß-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 ß-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 ß-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 ß-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.

Separation of clavulanic acid from fermented broth of amino acids by an aqueous two-phase system and ion-exchange adsorption.

The clavulanic acid is a substance which inhibits the ß-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.