Effect of beta-lactam antibiotics on lipid bilayer membranes. I. Penicillin antibiotics

Interaction between penicillins and model membrane systems, flat black bilayer lipid membranes (BLM) composed of vegetable or bacterial phospholipids was studied with an account of the complicated structure of bacterial cell membranes and possible presence in them of "pure" bilayer lipid areas. By their effect on electroconductivity of the BLM the antibiotics could be divided into three groups: those having no effect on the BLM electroconductivity at the maximum concentrations i.e. benzylpenicillin, carbenicillin, piperacillin (at pH 6.0 and 7.0) and ampicillin (at pH 6.0), those insignificantly changing electroconductivity of the BLM i.e. carfecillin and azlocillin and those having a significant effect on the BLM electroconductivity i.e. ampicillin N-acyl derivatives and 6-APA. The effect of ampicillin on the BLM conductivity markedly depended on the electrolite pH. The penicillins bound to the bilayer and induced changes in the transmembrane potential (evident from the changes in the second harmonic of the capacitive current) and the BLM elasticity-capacitance parameters (evident from the changes in the ratio of the amplitudes of the first and third harmonics). It was shown that all the penicillins penetrated through the BLM composed of either vegetable or bacterial phospholipids. The capacity for the transmembrane transfer without changing of the bilayer conductivity must be connected with the fact that the penetrating antibiotics did not induce any changes in the BLM structure. The effect on the conductivity probably depended in its turn on the form of the molecule and the ratio of the hydrophilic and hydrophobic parts in it.     

Sensitivity of pathogenic clostridia to antibiotics]

By the sensitivity levels of the gas infection causative agents, i. e. pathogenic Clostridia to antibiotics, the latter were conditionally divided into 4 groups. The 1st group included the most active antibiotics, such as tetracyclines,, penicillins, cephalosporins, rifampicin, 7-chlor-7-desoxylincomycin. Their minimum inhibitory and bactericidal concentrations did not usually exceed 2 gamma/ml. For most of the strains the inhibitory and bactericidal concentrations amounted to the tenth and hundredth fractions of gamma/ml. The antibiotics of the 2nd group, i. e. erythromycin, lincomycin,ristomycin and levomycetin inhibited multiplication and viability of pathogenic Clostridia in concentrations of 20 gamma/ml. Erythromycin was most active among them The 3rd group consisted of oleandomycin, novobiocin, geliomycin and azalomycin, the minimum inhibitory concentrations of them being 20 to 50 gamma/ml. The antibiotics of the 4th group, i. e. neomycin, monomycin, kanamycin, streptomycin, polymyxin and others affected pathogenic Clostridia at very high concentrations, amounting to the hundrenth and thousandth of gamma/ml.     

Enzymatic modifications of cephalosporins by cephalosporin acylase and other enzymes

Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and beta-lactamase are also covered.     

Enzymatic Modifications of Cephalosporins by Cephalosporin Acylase and Other Enzymes

ABSTRACT

Semisynthetic cephalosporins are important antibacterials in clinical practice. Semisynthetic cephalosporins are manufactured by derivatizing 7-aminocephalosporanic acid (7-ACA) and its desacetylated form. Microbial enzymes such as D-amino acid oxidase, glutaryl-7-ACA acylase and cephalosporin esterase are being used as biocatalysts for the conversion of cephalosporin C (CEPH-C) to 7-ACA and its desacetylated derivatives. Recent developments in the field of enzymatic modifications of cephalosporin with special emphasis on group of enzymes called as cephalosporin acylase is discussed in this review. Aspects related to screening methods, isolation and purification, immobilization, molecular cloning, gene structure and expression and protein engineering of cephalosporin acylases have been covered. Topics pertaining to enzymatic modifications of cephalosporin by D-amino acid oxidase, cephalosporin methoxylase and β -lactamase are also covered.     

头孢菌素C生物合成调控研究进展北大核心CSCD

头孢菌素C由丝状真菌顶头孢霉产生,属于β-内酰胺类抗生素。其经改造后的7-氨基头孢烷酸是头孢类抗生素的重要中间体。头孢类抗生素在国内外抗生素市场中占有巨大的份额,是临床上的主要抗感染药物。随着分子生物学的发展,头孢菌素C的生物合成途径已基本阐明。为提高头孢菌素C的产量和降低生产成本,越来越多的研究者开始关注其较为精细、复杂的调控机制。本文重点对头孢菌素C生物合成及其调控机制的最新进展进行了简述,希望为今后头孢菌素C生产菌株的菌种改造和传统产业的升级换代提供一定的借鉴。     

Comparative characterization of new glutaryl 7-ACA and cephalosporin C acylases

We performed a comparative characterization of three new cephalosporin acylases which were prepared from E. coli recombinant strains and found originally from Pseudomonas sp. A14, Bacillus laterosporus J1 and Pseudomonas diminuta N176. Both A14 and N176 acylases consisted of two non-identical subunits (α, β) whose molecular weights were 28,000 (α), 61,000 (β) and 26,000 (α), 58,000 (β), respectively, whereas J1 acylase consisted of a single peptide with molecular weight of 70,000. The maximum specific activities of A14, J1 and N176 acylases for glutaryl 7-ACA were 7.1, 5.3 and 100 units/mg, respectively, and that of N176 acylase for cephalosporin C was 3.1 units/mg. The K_m values of glutaryl 7-ACA for A14, J1 and N176 acylases were 2.1, 3.2 and 2.6 mM, respectively, and that of cephalosporin C for N176 acylase was 4.8 mM. A14, J1 and N176 acylases exhibited differential activities for cephalosporins having an aliphatic dicarboxylic acid in the acyl side chain and only N176 acylase showed an activity for cephalosporin C. N176 acylase as well as A14 acylase also showed a weak activity for a cephalosporin derivative having a heterocyclic carboxylic acid in the side chain. A14, J1 and N176 acylases catalyzed the reverse reaction to synthesize glutaryl 7-ACA from 7-ACA and glutaric acid, although the rate of the synthesis was 10 to 10⁵ fold slower than that of hydrolysis. The activities of the cephalosporin acylases were considerably inhibited by the reaction products, 7-ACA and glutaric acid. The types of the inhibition by 7-ACA and glutaric acid were both competitive. A14, J1 and N176 acylases were thermostable, their residual activities exceeding more than 90% after treatment at 50°C for 1 h at their optimal pHs.     

Modifying the substrate specificity of penicillin G acylase to cephalosporin acylase by mutating active-site residues

The penicillin G acylase (PGA) and cephalosporin acylase (CA) families, which are members of the N-terminal (Ntn) hydrolases, are valuable for the production of backbone chemicals like 6-aminopenicillanic acid and 7-aminocephalosporanic acid (7-ACA), which can be used to synthesize semi-synthetic penicillins and cephalosporins, respectively. Regardless of the low sequence similarity between PGA and CA, the structural homologies at their active-sites are very high. However, despite this structural conservation, they catalyze very different substrates. PGA reacts with the hydrophobic aromatic side-chain (the phenylacetyl moiety) of penicillin G (PG), whereas CA targets the hydrophilic linear side-chain (the glutaryl moiety) of glutaryl-7-ACA (GL-7-ACA). These different substrate specificities are likely to be due to differences in the side-chains of the active-site residues. In this study, mutagenesis of active-site residues binding the side-chain moiety of PG changed the substrate specificity of PGA to that of CA. This mutant PGA may constitute an alternative source of engineered enzymes for the industrial production of 7-ACA.     

Recombinant microorganisms for industrial production of antibiotics

The enhancement of industrial antibiotic yield has been achieved through technological innovations and traditional strain improvement programs based on random mutation and screening. The development of recombinant DNA techniques and their application to antibiotic producing microorganisms has allowed yield increments and the design of biosynthetic pathways giving rise to new antibiotics. Genetic manipulations of the cephalosporin producing fungus Cephalosporium acremonium have included yield improvements, accomplished increasing biosynthetic gene dosage or enhancing oxygen uptake, and new biosynthetic capacities as 7-aminocephalosporanic acid (7-ACA) or penicillin G production. Similarly, in Penicillium chrysogenum, the industrial penicillin producing fungus, heterologous expression of cephalosporin biosynthetic genes has led to the biosynthesis of adipyl-7-aminodeacetoxycephalosporanic acid (adipyl-7-ADCA) and adipyl-7-ACA, compounds that can be transformed into the economically relevant 7-ADCA and 7-ACA intermediates. Escherichia coli expression of the genes encoding D-amino acid oxidase and cephalosporin acylase activities has simplified the bioconversion of cephalosporin C into 7-ACA, eliminating the use of organic solvents. The genetic manipulation of antibiotic producing actinomycetes has allowed productivity increments and the development of new hybrid antibiotics. A legal framework has been developed for the confined manipulation of genetically modified organisms. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 216-226, 1997.     

Selective reminiscences of beta-lactam antibiotics: early research on penicillin and cephalosporins

The discovery, made in Oxford, that crude penicillin could cure systemic and life-threatening bacterial infections was followed by attempts to purify penicillin, to determine its structure and then to produce it by total chemical synthesis. The beta-lactam structure of the molecule, first proposed in October 1943, was a source of controversy until 1945. However, no useful chemical synthesis was achieved and fermentation became the commercial source of the antibiotic. In 1953, one of the products of a Cephalosporium sp. from Sardinia was shown to be a new and hydrophilic penicillin (penicillin N). This was contaminated with a substance having the same side-chain but a characteristic absorption spectrum. The latter, cephalosporin C, showed antibacterial activity but was not inactivated by a penicillinase. The determination of its beta-lactam structure and isolation of its nucleus enabled pharmaceutical companies to produce many semisynthetic cephalosporins. A new tripeptide was later found to be an intermediate in the biosynthesis of both penicillin N and cephalosporin C, and this was followed by the complete elucidation of the biosynthetic pathways leading to these compounds and to benzylpenicillin.     

Physical Properties and Kinetic Behavior of a Cephalosporin Acetylesterase Produced by Bacillus subtilis

An esterase that deacetylates cephalosporins was recovered from the supernatant of a Bacillus subtilis culture. It was partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme had a temperature optimum between 40 and 50 C and a pH optimum of 7.0. The molecular weight was estimated by gel filtration to be 190,000. The enzyme was very stable and retained greater than 80% of its activity after storage in solution at 25 C for 1 month. The esterase exhibited Michaelis-Menton kinetics with the substrates 7-aminocephalosporanic acid (7-ACA) and 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin); the K(m) values were 2.8 X 10(-3) and 8.3 X 10(-3) M, respectively. The products of 7-ACA deacetylation were weak competitive inhibitors, and a K(i) value of 5.0 X 10(-2) M was determined for acetate and of 3.6 X 10-2 M for deacetyl-7-ACA. Weak product inhibition did not prevent the deacetylation reaction from going to completion. A 5-mg/ml solution of partially purified esterase completely hydrolyzed (greater than 99.5%) a 24-mg/ml solution of 7-ACA in 3 h. Because of the kinetic properties and excellent stability, this enzyme may be useful in an immobilized form to prepare large quantities of deacetylated cephalosporin derivatives.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

The Pharmacology of Semisynthetic Antibiotics

The chemical structures and reactions of penicillins and cephalosporins are reviewed in relation to their effects upon pharmacodynamic properties. The reactive betalactam ring is common to all penicillins and cephalosporin C analogues. This ring opens during acylation of the bacterial wall-building enzymes, but previous opening of the ring by acid or beta-lactamases destroys antibiotic activity.Semisynthetic substitutions may protect the ring by steric hindrance; this may actually inactivate certain penicillinases, so that resistant penicillins may potentiate penicillin G in some circumstances. However, the protective substitutions reduce the intrinsic activity of the synthetic penicillins themselves. Other properties which are affected include absorption, protein-binding, excretion, and possible allergenicity of the drugs. Effects on antibacterial spectrum may possibly be secondary to alteration of lipid solubility.     

Biosynthesis of peptidoglycan in Pseudomonas aeruginosa. 2. Mode of action of beta-lactam antibiotics.

The intrinsic effect of various beta-lactam antibiotics on the biosynthesis of peptidoglycan of Pseudomonas aeruginosa X-48 was investigated. Most of the cephalosporins and penicillins tested already at 0.5 microgram/ml strongly inhibited (a) the incorporation of nascent peptidoglycan into the detergent-insoluble fraction (greater than 75%), (b) the formation of peptide crosslinkages (greater than 60%) and (c) the activity of the DD-carboxypeptidase and partially that of the transpeptidase (approximately 90% and approximately 40% respectively). Another group of beta-lactum drugs did not inhibit incorporation into the material insoluble in sodium dodecylsulfate, the formation of peptide crosslinkages nor transpeptidase activity. They only partially inhibited the activity of the DD-carboxypeptidase--endopeptidase system (40--50% at 0.5 microgram/ml). The results obtained differ from those of Presslitz and Ray [Antimicrob, Agents Chemother. 7, 578--581 (1975)] and show some resemblance to the effects of beta-lactams on the biosynthesis of Escherichia coli peptidoglycan.     

Lysine epsilon-aminotransferase, the initial enzyme of cephalosporin biosynthesis in actinomycetes

Streptomyces clavuligerus, Streptomyces lipmanii and Nocardia (formerly Streptomyces) lactamdurans are Gram-positive mycelial bacteria that produce medically important beta-lactam antibiotics (penicillins and cephalosporins including cephamycins) that are synthesized through a series of reactions starting from lysine, cysteine and valine. L-lysine epsilon-aminotransferase (LAT) is the initial enzyme in the two-step conversion of L-lysine to L-alpha-aminoadipic acid, a specific precursor of all penicillins and cephalosporins. Whereas S. clavuligerus uses LAT for cephalosporin production, it uses the cadaverine pathway for catabolism when lysine is the nitrogen source for growth. Although the cadaverine path is present in all examined streptomycetes, the LAT pathway appears to exist only in beta-lactam-producing strains. Genetically increasing the level of LAT enhances the production of cephamycin. LAT is the key rate-limiting enzyme in cephalosporin biosynthesis in S. clavuligerus strain NRRL 3585. This review will summarize information on this important enzyme.     

Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-alpha-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50betaM/Y149alphaK/F177betaG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.     

Achievements and problems from the view of a physician

Penicillin made possible the cure of many common, and also the most serious, infections, such as meningococcal meningitis and bacterial endocarditis, often with few or no sequelae. Endocarditis had been invariably fatal. Semisynthetic penicillins added new dimensions of convenience of administration and a broader spectrum in the presence of many beta-lactamases. A quantum step forward was permitted by the derivatives of cephalosporin C. Specific clinical advances were (1) the opportunity to use these in some penicillin-allergic patients, (2) activity against wider range of Gram-negative bacilli, (3) activity against Bacteroides fragilis (cefoxitin), (4) more complete renal excretion after oral cephalosporins than with oral penicillins, and (5) delayed renal excretion. Major remaining problems limiting beta-lactam use are (1) allergy, (2) resistant organisms, (3) relatively poor entry into the cerebrospinal fluid (especially of cephalosporins, (4) some nephrotoxicity, (5) local irritation of veins and tissues during administration, and (6) poor results in patients with agranulocytosis.     

Industrial production of beta-lactam antibiotics

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.     

Gene clusters for beta-lactam antibiotics and control of their expression: why have clusters evolved, and from where did they originate?

While beta-lactam compounds were discovered in filamentous fungi, actinomycetes and gram-negative bacteria are also known to produce different types of beta-lactams. All beta-lactam compounds contain a four-membered beta-lactam ring. The structure of their second ring allows these compounds to be classified into penicillins, cephalosporins, clavams, carbapenens or monobactams. Most beta-lactams inhibits bacterial cell wall biosynthesis but others behave as beta-lactamase inhibitors (e.g., clavulanic acid) and even as antifungal agents (e.g., some clavams). Due to the nature of the second ring in beta-lactam molecules, the precursors and biosynthetic pathways of clavams, carbapenems and monobactams differ from those of penicillins and cephalosporins. These last two groups, including cephamycins and cephabacins, are formed from three precursor amino acids that are linked into the alpha-aminoadipyl-L-cysteinyl-D-valine tripeptide. The first two steps of their biosynthetic pathways are common. The intermediates of these pathways, the characteristics of the enzymes involved, the lack of introns in the genes and bioinformatic analysis suggest that all of them should have evolved from an ancestral gene cluster of bacterial origin, which was surely transferred horizontally in the soil from producer to non-producer microorganisms. The receptor strains acquired fragments of the original bacterial cluster and occasionally inserted new genes into the clusters, which once modified, acquired new functions and gave rise to the final compounds that we know. When the order of genes in the Streptomyces genome is analyzed, the antibiotic gene clusters are highlighted as gene islands in the genome. Nonetheless, the assemblage of the ancestral beta-lactam gene cluster remains a matter of speculation.     

Effect of tetracycline group antibiotics on the barrier properties of the small intestine epithelium]

The effect of 5 tetracyclines on the barrier and transport properties of the small intestine epithelium was studied. The barrier properties were estimated by a change in the ionic selectivity and conductivity of the epithelium, as well as by enterocyte linkage. The current of the short circuit served the characteristics of the Na transport system state. Dimethylchlortetracycline, methacycline and oxytetracycline in concentrations of 10(-7) g/ml decreased the epithelium conductivity and increased the cell linkage. Tetracycline and methacycline in concentrations of 10(-9) g/ml had an analogous effect. The effect was observed 10--20 minutes after the start of incubation with the substance. No effect on the current of the short circuit was observed within the concentrations of 10(-4) to 10(-9) g/ml. It is suggested that the decreased conductivity and increased linkage are due to adsorption of the tetracycline molecules in the region of close cell contacts.     

Inhibitors of metallo-beta-lactamase generated from beta-lactam antibiotics

The resistance of bacteria to the normally lethal action of beta-lactam antibiotics is largely due to the production of beta-lactamases that catalyze the hydrolysis of the beta-lactam. One class of these enzymes is a zinc-dependent metallo-beta-lactamase for which there are no clinically available inhibitors. The hydrolysis of cephalosporin beta-lactam antibiotics generates dihydrothiazines which subsequently undergo isomerization at C6 by C-S bond cleavage and through the intermediacy of a thiol. These thiols can be trapped by the beta-lactamase from Bacillus cereus, causing inhibition of the enzyme. The rate of production of the thiol corresponds to the rate of inhibition, and the inhibition constants are in the micromolar range but vary with the nature of the cephalosporin derivative. NMR studies have identified the structure of the thiols causing inhibition and also show that the thiol binds to the zinc ion, which in turn perturbs the metal-bound histidines. Inhibition is slowly removed as the thiol becomes oxidized or undergoes further degradation. The thiol intermediate generated from cephalothin is a slow binding inhibitor. There is no observed inhibition from the analogous degradation products from penicillins.