Phenoxymethylpenicillin amidohydrolases from Penicillium chrysogenum.

A phenoxymethylpenicillin amidohydrolase which hydrolyses phenoxymethylpenicillin to 6-aminopenicillanic acid (6-APA) has been isolated from two species of Penicillium chrysogenum. The amidohydrolase had a molecular mass of approx. 42 kDa. Its activity with benzylpenicillin as substrate was only 1.5% of that with phenoxymethylpenicillin and it was inhibited by its products. No penicillin formation from 6-APA and phenoxyacetyl or phenylacetyl coenzyme A was observed. The enzyme is thus distinct from the phenylacetyl coenzyme A:6-APA acyltransferase, which also has amidohydrolase activity and is involved in the final stages of the biosynthesis of penicillins.

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans.

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.

Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.

Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.

Cephalosporins 1945-1986.

In 1945, after penicillin had been introduced into medicine, an antibiotic-producing species of Cephalosporium was isolated from a sewage outfall in Sardinia. Four years later in Oxford, this organism was found to produce several antibiotics, one of which was a penicillin with a new side-chain, penicillin N. During a chemical study in 1953, this penicillin was shown to be contaminated with a second substance, cephalosporin C, which contained a beta-lactam ring but was resistant to hydrolysis by a penicillinase (beta-lactamase). At that time, penicillinase-producing Staphylococci were causing a serious problem in hospitals. The isolation of the nucleus of cephalosporin C (7-ACA) enabled pharmaceutical manufacturers to produce many thousands of cephalosporins, some of which have been effective in the treatment of serious infections by a number of Gram-positive and Gram-negative bacteria. The cephalosporins, like the newer penicillins, have a very low toxicity and have greatly extended the range of chemotherapy. New, sensitive screening methods have revealed further families of clinically useful substances that contain a reactive beta-lactam ring. Genetic engineering has now begun to throw light on the nature of the enzymes that are involved in the biosynthesis of penicillins and cephalosporins, and x-ray crystallography may soon provide detailed 3-dimensional pictures of some of the bacterial enzymes with which the active beta-lactam ring reacts. Rational approaches to the production and design of new and potentially useful compounds may then be within sight.

A spectroscopic study of metal ion and ligand binding to beta-lactamase II.

beta-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted beta-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to beta-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4 degrees C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 microM (pH 6.0, 30 degrees C, 1 M NaCl) for the dissociation constant of Zn(II). The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.

Purification and characterization of cloned isopenicillin N synthetase.

Isopenicillin N synthetase (IPS) cloned from Cephalosporium acremonium has been isolated from transformed Escherichia coli and purified to homogeneity. The resulting, abundant, recombinant protein, whilst undergoing slightly different N-terminal processing to that observed for the fungally-derived protein, has identical kinetics for the conversion of LLD-aminoadipoyl-cysteinyl-valine to isopenicillin N. Recombinant IPS converts analogue substrates into unusual beta-lactam antibiotics in exactly the same way as the fungal protein.

A retrospective view of beta-lactamases.

The discovery of a penicillinase (later shown be a beta-lactamase) 50 years ago in Oxford came from the thought that the resistance of many Gram-negative bacteria to Fleming's penicillinase might be due to their production of a penicillin-destroying enzyme. The emergence of penicillinase-producing staphylococci in the early 1950s, particularly in hospitals, raised the question whether the medical value of penicillin would decline. The introduction of new semi-synthetic penicillins and cephalosporins in the 1960s began to reveal many beta-lactamases distinguishable by their different substrate profiles. In this period it was established that genes encoding beta-lactamases from Gram-negative bacilli could be carried from one organism to another on plasmids and also that penicillin inhibited a transpeptidase involved in bacterial cell wall synthesis. During the last two decades a number of these enzymes have been purified and the genes encoding them have been cloned. Much has now been learned, with the aid of powerful modern techniques, about their structures, their active sites, their relationship to penicillin-sensitive proteins in bacteria and to their likely evolution. Further knowledge may contribute to a more rational approach to chemotherapy in this area. Experience suggests that a need for new substances will continue.

A glimpse of the early history of the cephalosporins.

The cephalosporins, like the penicillins, came from research that was partly academic but that led to results which found application in medicine. A number of events followed the isolation of a Cephalosporium in Sardinia in 1945. Research at Oxford resulted in the discovery of cephalosporin C in 1953, in the elucidation of its structure in 1959, and in the determination of many of its characteristic properties. Further work in the United States opened the way to large-scale production of a series of semisynthetic cephalosporins. A change in the attitude of the Government toward the application of academic research in the United Kingdom and the establishment of a National Research Development Corporation were responsible for certain differences between the commercial development of the cephalosporins and that of penicillin.

Antimicrobial activities and antagonists of bacilysin and anticapsin.

The dipeptide antibiotic bacilysin is active against a wide range of bacteria and against Candida albicans. Its C-terminal amino acid, anticapsin, is a very poor antibacterial agent. The activities of both substances are strongly dependent on the nature of the culture medium. In a minimal medium the minimum inhibitory concentration for bacilysin with E. coli B is 10(-3) mug ml(-1). The action of bacilysin amino acids. With several bacteria, bacilysin-resistant mutants are found in unusually large numbers. It is suggested that peptide and amino acid transport systems play a role in these phenomena. The antimicrobial action of bacilysin is also inhibited by glucosamine and N-acetylglucosamine. This antibiotic may therefore interfere with glucosamine synthesis and thus with the synthesis of microbial cell walls.

Conformational changes in the extracellular beta-lactamase I from Bacillus cereus 569/H/9.

1. The thermal denaturation and precipitation of beta-lactamase I from Bacillus cereus 569/H/9 at 60 degrees C are reversible, a soluble and almost fully active enzyme being obtained after solution of the precipitate in 5m-guanidinium chloride or 8m-urea and subsequent removal of the denaturing agent. 2. Inactivation of beta-lactamase I occurs rapidly between 50 degrees and 55 degrees C and is shown by circular-dichroism spectra to be accompanied by an extensive conformational change. 3. A change to a different conformation occurs in 6m-urea. This change is also reversible; refolding with almost complete recovery of enzymic activity occurs within 5min of dilution of the denaturing agent. 4. Inactivation of beta-lactamase I at pH3.0 and 11.0 is also associated with conformational changes, since a proportion of the lost activity is recovered within 5min of adjustment of the pH to 7.0.

Separation, purification and properties of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9.

1. A procedure was devised which is suitable for the isolation of beta-lactamase I and beta-lactamase II from Bacillus cereus 569/H/9 on a large scale. After adsorption on to Celite both enzymes were eluted in good yield and separated by chromatography on Sephadex CM-50. 2. beta-Lactamase I was separated into three main components by isoelectric focusing and into two components by chromatography. 3. The Zn(2+)-requiring beta-lactamase II obtained by this procedure had a lower molecular weight (22000) than beta-lactamase I (28000) and also differed from the latter in containing one cysteine residue. 4. The beta-lactamase II contained no carbohydrate, but showed the thermostability of the enzyme isolated earlier as a protein-carbohydrate complex. 5. Amino acid analyses and tryptic-digest ;maps' indicate that some degree of homology between beta-lactamase I and beta-lactamase II is possible, but that beta-lactamase I is not composed of the entire sequence of beta-lactamase II together with an additional peptide fragment. 6. A 6-methylpenicillin and a 7-methylcephalosporin showed much lower affinities for both enzymes than did penicillins and cephalosporins themselves.

Metal cofactor requirements of beta-lactamase II.

1. The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn(2+)-containing enzyme. 2. Removal of Zn(2+) led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn(2+) by Co(2+), Cd(2+), Mn(2+) or Hg(2+) gave enzymes with significant, but lower, beta-lactamase activity. No activity was detected in the presence of Cu(2+), Ni(2+), Mg(2+) or Ca(2+). 4. Equilibrium dialysis indicated that the enzyme had at least two Zn(2+) binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn(2+) indicated that activity paralleled binding of Zn(2+) to the site of highest affinity. 5. Replacement of Zn(2+) by Co(2+) and Cd(2+) gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured beta-lactamase II with Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce beta-lactamase II activity in high yield.

Beta-lactamases from Yersinia enterocolitica.

Two beta-lactamases, A and B, have been shown to be present in a strain of Yersinia enterocolitica (w222). Beta-Lactamase A hydrolyses a variety of penicillins and cephalosporins. This enzyme is sensitive to thiol reagents, is only partially inhibited by 0-1 mM-cloxacillin and has a molecular weight of approximatley 20,000.beta-Lactamase B shows strong cephalosporinase activity but does not hydrolyse some of the penicillins. It is more resistant than beta-lactamase A to thiol reagents, is completely inhibited by 0-1 mM-cloxacillin and has a molecular weight of about 34,000. With cephaloridine as a substrate, which is readily hydrolysed by both enzymes, about 85% of the total activity of a cell extract is due to beta-lactamase A and 15% to B. Addition of 6-aminopenicillanic acid to the culture during growth results in a 2-to4-fold selective increase in the amount of beta-lactamase B. Two beta-lactamases similar to enzymes A and B have been found in five other strains of Y. enterocolitica. In contrast, only one beta-lactamase, similar to enzyme B, has been detected in a different strain of Y. enterocolitica (H66), which is abnormal in that it is sensitive to ampicillin. Addition of 6-aminopenicillanic acid to cultures of this strain results in an 8-to 10-fold increase in beta-lactamase production.

The conversion of cephalosporins to 7 alpha-methoxycephalosporins by cell-free extracts of Streptomyces clavuligerus.

In the presence of S-adenosylmethionine, 2-oxoglutarate, Fe2+ and a reducing agent, cell-free extracts of Streptomyces clavuligerus convert cephalosporin C and O-carbamoyldeacetylcephalosporin C into 7 alpha-methoxy derivatives. No synthesis of a 7 alpha-methoxy derivative of deacetylcephalosporin C was detected in the system used, and the 7 alpha-methoxy derivative of deacetoxycephalosporin C was produced only in relatively small amounts. It appears that the 7 alpha-methoxy group is introduced after the cephalosporin ring system has been formed and that its introduction may represent the final step in a biosynthetic pathway.