Interactions of isopenicillin N synthase with cyclopropyl-containing substrate analogues reveal new mechanistic insight

Isopenicillin N synthase (IPNS), a non-heme iron oxidase central to penicillin and cephalosporin biosynthesis, catalyzes an energetically demanding chemical transformation to produce isopenicillin N from the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV). We describe the synthesis of two cyclopropyl-containing tripeptide analogues, delta-(l-alpha-aminoadipoyl)-l-cysteinyl-beta-methyl-d-cyclopropylglycine and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-cyclopropylglycine, designed as probes for the mechanism of IPNS. We have solved the X-ray crystal structures of these substrates in complex with IPNS and propose a revised mechanism for the IPNS-mediated turnover of these compounds. Relative to the previously determined IPNS-Fe(II)-ACV structure, key differences exist in substrate orientation and water occupancy, which allow for an explanation of the differences in reactivity of these substrates.     

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase,that mediates the first committed step in penicillin biosynthesis,is a cytosolic enzyme

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.     

Thioesterase domain of delta-(l-alpha-Aminoadipyl)-l-cysteinyl-d-valine synthetase: alteration of stereospecificity by site-directed mutagenesis

The carboxy-terminal thioesterase domain of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase catalyzes the hydrolytic release of the tripeptide product (LLD-ACV). By site-directed mutagenesis an S3599A change was introduced into the highly conserved GXSXG motif, resulting in a more than 95 % decrease of penicillin production. Purification of the modified multienzyme showed surprisingly only a 50 % reduction of the peptide formation rate, with the stereoisomer delta-(l-alpha-aminoadipyl)-l-cysteinyl-l-valine (LLL-ACV) as the dominating product. Thioesterases of ACV synthetases differ from other thioesterases integrated in non-ribosomal peptide synthetases in their direct association with an epimerase domain, and their respective GXSXG-seryl residue is apparently not essential in acyl transfer leading to peptide release. Instead, this motif may be involved in the control of tripeptide epimerization by selection of the isomer to be released, and the construct supports the presence of LLL-ACV as an intermediate in penicillin biosynthesis.     

Structural studies on the reaction of isopenicillin N synthase with the truncated substrate analogues delta-(L-alpha-aminoadipoyl)-L-cysteinyl-glycine and delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-alanine

Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversion of the tripeptide delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to bicyclic isopenicillin N (IPN), concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues delta-(l-alpha-aminoadipoyl)-l-cysteinyl-glycine (ACG) and delta-(l-alpha-aminoadipoyl)-l-cysteinyl-d-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substrate cysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for the anaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of the dioxygen analogue nitric oxide. The overall protein structures are very similar to those of the corresponding IPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the active site iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of this substrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional water ligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex. ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. The addition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These results further demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymes and provide insight into the delicate balance between hydrophilic-hydrophobic interactions and steric effects in the IPNS active site.     

Delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. Molecular characterization of the acvA gene encoding the first enzyme of the penicillin biosynthetic pathway

The Aspergillus nidulans gene (acvA) encoding the first catalytic steps of penicillin biosynthesis that result in the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), has been positively identified by matching a 15-amino acid segment of sequence obtained from an internal CNBr fragment of the purified amino-terminally blocked protein with that predicted from the DNA sequence. acvA is transcribed in the opposite orientation to ipnA (encoding isopenicillin N synthetase), with an intergenic region of 872 nucleotides. The gene has been completely sequenced at the nucleotide level and found to encode a protein of 3,770 amino acids (molecular mass, 422,486 Da). Both fast protein liquid chromatography and native gel estimates of molecular mass are consistent with this predicted molecular weight. The enzyme was identified as a glycoprotein by means of affinity blotting with concanavalin A. No evidence for the presence of introns within the acvA gene has been found. The derived amino acid sequence of ACV synthetase (ACVS) contains three homologous regions of about 585 residues, each of which displays areas of similarity with (i) adenylate-forming enzymes such as parsley 4-coumarate-CoA ligase and firefly luciferase and (ii) several multienzyme peptide synthetases, including bacterial gramicidin S synthetase 1 and tyrocidine synthetase 1. Despite these similarities, conserved cysteine residues found in the latter synthetases and thought to be essential for the thiotemplate mechanism of peptide biosynthesis have not been detected in the ACVS sequence. These observations, together with the occurrence of putative 4'-phosphopantetheine-attachment sites and a putative thioesterase site, are discussed with reference to the reaction sequence leading to production of the ACV tripeptide. We speculate that each of the homologous regions corresponds to a functional domain that recognizes one of the three substrate amino acids.     

Enzymatic characterisation of the multifunctional enzyme delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Streptomyces clavuligerus.

delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-alpha-aminoadipic-acid-dependent ATP/PPi exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with alpha-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid.     

A coupled enzyme assay for isopenicillin N synthetase

The development of a coupled enzyme assay for the determination of isopenicillin N synthetase activity in purified extracts from Cephalosporium acremonium was described. Isopenicillin N formed from its precursor, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), by the synthetase was hydrolyzed by beta-lactamase I to the corresponding penicilloic acid. Automatic titration of the acid with standard sodium hydroxide delivered by a pH-stat gave a continuous plot of product formed vs time. This assay has been used in kinetic studies and to determine the effects of pH, ionic strength, and temperature on the enzyme's activity.     

A study of the biosynthesis of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in a beta-lactam-negative mutant of Cephalosporium acremonium.

A cell-free extract of Cephalosporium acremonium (Takeda N-2) was obtained that synthesized the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and also the dipeptide delta-(L-alpha-aminoadipyl)-L-cysteine from the corresponding L-amino acids.     

Spectroscopic studies of isopenicillin N synthase. A mononuclear nonheme Fe2+ oxidase with metal coordination sites for small molecules and substrate

The nonheme iron oxidase isopenicillin N synthase catalyzes the formation of two new internal bonds in the tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to form the beta-lactam and thiazolidine rings of isopenicillin N. Concomitantly, O2 is reduced to 2 H2O. The recombinant enzyme from Cephalosporium acremonium (Mr = 38,400), expressed as an apoenzyme in Escherichia coli, binds 1 g atom of Fe2+/mol of enzyme to reconstitute full activity. M?ssbauer spectra of the 57Fe-enriched enzyme exhibit parameters (delta = 1.30 mm/s, delta EQ = 2.70 mm/s) which unambiguously show that the active site iron is high spin Fe2+. Anaerobic binding of ACV causes a substantial decrease in the isomer shift parameter delta (delta = 1.10 mm/s, delta EQ = 3.40 mm/s) showing that the substrate perturbs the iron site and makes its coordination environment much more covalent. Nitric oxide (NO) binds to the EPR silent active site iron to give an EPR active species (g = 4.09, 3.95, 2.0; S = 3/2) similar to those of the nitrosyl complexes of many other mononuclear Fe2+-containing enzymes. The rhombicity of the EPR spectrum is increased (g = 4.22, 3.81, 1.99) by anaerobic addition of ACV suggesting that the substrate binds to or near the iron without displacing NO. Interestingly, the enzyme.ACV.NO complex displays an optical spectrum similar to that of ferric rubredoxin in which the iron has only thiol coordination. This suggests that the Fe2+ of the enzyme.ACV.NO complex acquires Fe3+ character and that the cysteinyl thiol moiety of ACV coordinates to the iron. Similar substrate thiol coordination to the iron of the enzyme.ACV complex is the most probable explanation for the large decrease in isomer shift observed. These results provide the first evidence for the direct involvement of iron in this unique O2-dependent reaction and suggest novel roles for iron and oxygen in biological catalysis.     

Purification and characterization of the isopenicillin N synthase of Streptomyces lactamdurans

The isopenicillin N synthase (cyclase) of Streptomyces lactamdurans (syn. Nocardia lactamdurans) has been purified to near homogeneity as judged by SDS-PAGE and isoelectric focusing. This enzyme catalyses the oxidative cyclization of the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The enzyme required DTT, Fe2+ and oxygen and it was greatly stimulated by ascorbic acid. It was strongly inhibited by Co2+, Zn2+ and Mn2+. Optimal pH and temperature were 7.0 and 25 degrees C (with the assay conditions used), respectively. The apparent Km of isopenicillin N synthase for delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine was 0.18 mM. The enzyme is a monomer with an Mr of 26,500 +/- 1000 and a pI of 6.55.     

Kinetic analysis of cephalosporin biosynthesis in Streptomyces clavuligerus

A kinetic model describing the cephalosporin biosynthesis in Streptomyces clavuligerus was developed. Using previously reported kinetic data of biosynthetic enzymes, we examined the kinetics of cephalosporin production. The predicted time profile of the specific production rate during a batch culture parallels that of experimental observation. Sensitivity analysis reveals that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase is the rate-limiting enzyme. The effect of amplifying ACV synthetase on the specific production rate was analyzed theoretically. Increasing ACV synthetase enhances the production rate initially until ACV synthetase enhances the production rate initially until deacetocycephalosporin C hydroxylase becomes rate-limiting. Such kinetic analysis can provide a rational basis for modifying the biosynthetic machinery of cephalosporin through gene cloning.     

Site-directed mutagenesis of arginine-89 supports the role of its guanidino side-chain in substrate binding by Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) catalyses a key step in the penicillin and cephalosporin biosynthetic pathway which involves the oxidative cyclisation of the acyclic peptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N. Based on crystallographic evidence from the Aspergillus nidulans IPNS crystal structure complexed with the substrate ACV (Roach et al. (1997) Nature 387, 827-830), we were able to provide mutational evidence for the critical involvement of the conserved R-X-S motif in ACV binding in IPNS. The crystal structure further implicated arginine-87 in the binding of the aminoadipyl portion of ACV. Thus, in this study, the site-directed mutagenesis of the corresponding arginine-89 in Cephalosporium acremonium IPNS (cIPNS) was performed to ascertain its role in cIPNS. Alteration of arginine-89 to five amino acids from different amino acid groups, namely lysine, serine, alanine, aspartate and leucine, was performed and no activity was detected in all the mutants obtained when enzyme bioassays were performed. Furthermore, the solubility of the mutants was considerably lower than the wild-type cIPNS after expression at 37 degrees C, but could be recovered when the expression temperature was lowered to 25 degrees C. This suggests that arginine-89 could be critical for the activity of cIPNS due to its involvement in ACV binding and the solubility of wild-type enzyme.     

Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.     

Low-molecular-weight thiols in streptomycetes and their potential role as antioxidants.

The intracellular low-molecular-weight thiols present in five gram-positive Streptomyces species and one Flavobacterium species were analyzed by high-performance liquid chromatography after fluorescence labeling with monobromobimane. Bacteria were chosen to include penicillin and cephalosporin beta-lactam producers and nonproducers. No significant amount of glutathione was found in any of the streptomycetes. Major intracellular thiols in all strains examined were cysteine, coenzyme A, sulfide, thiosulfate, and an unknown thiol designated U17. Those streptomycetes that make beta-lactam antibiotics also produce significant amounts of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV), a key intermediate in their biosynthesis. In Streptomyces clavuligerus, a potent producer of beta-lactams, the level of ACV was low during the early phase of growth and increased rapidly toward the end of exponential growth, paralleling that of antibiotic production. These and other observations indicate that ACV does not function as a protective thiol in streptomycetes. U17 may have this role since it was the major thiol in all streptomycetes and appeared to occur at levels about 10-fold higher than those of the other thiols measured, including ACV. Purification and amino acid analysis of U17 indicated that it contains cysteine and an unusual amine that is not one of the common amino acids. This thiol is identical to an unknown thiol found previously in Micrococcus roseus and Streptomyces griseus. A high level of ergothioneine was found in Streptomyces lactamdurans, and several unidentified thiols were detected in this and other streptomycetes.     

Mutational analysis of tyrosine-191 in the catalysis of Cephalosporium acremonium isopenicillin N synthase

Isopenicillin N synthase (IPNS) is a key enzyme responsible for the catalytic conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. The Aspergillus nidulans IPNS crystal structure implicated amino acid residues tyrosine-189, arginine-279, and serine-281 in the substrate-binding of the valine carboxylate portion of ACV via hydrogen bonds. In previous reports, we provided mutational evidence for the critical involvement of the corresponding arginine-281 and serine-283, which constitute a conserved R-X-S motif, for the catalysis of Cephalosporium acremonium IPNS (cIPNS). In this study, we report the site-directed mutagenesis of the corresponding tyrosine-191 in cIPNS to four amino acids from different amino acid groups, namely, phenylalanine, serine, histidine, and aspartate. The mutants Y191F, Y191H, and Y191R respectively yielded specific activities at levels of 3, 8.6, and 18.8% relative to the wild-type when enzyme bioassays were performed using purified protein fractions. These results were surprising, as previous mutational analyses involving arginine-281 and serine-283 resulted in non-measurable specific activities, thus suggesting that tyrosine-191 is important but not critical for the activity of cIPNS due to its involvement in ACV binding. Hence, it is likely that tyrosine-191 is the least critical of the three residues involved in binding the ACV valine carboxylate moiety.     

Purification from Cephalosporium acremonium of the initial enzyme unique to the biosynthesis of penicillins and cephalosporins

The stability of the unstable enzyme, delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase from Cephalosporium acremonium C-10, was increased 10-fold which facilitated its purification. The active enzyme was purified over 100 fold to electrophoretic homogeneity by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration and hydrophobic interaction chromatography. It appears to have a minimal size of 360 kDa based on SDS-polyacrylamide gel electrophoresis.     

delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Aspergillus nidulans. The first enzyme in penicillin biosynthesis is a multifunctional peptide synthetase

A multienzyme catalyzing the formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the first free intermediate in penicillin biosynthesis, was detected in an assay measuring the formation of tripeptide from L-[U-14C]valine in the presence of L-alpha-aminoadipic acid, L-cysteine, ATP, Mg2+ ions, and dithioerythritol. Enzyme was extracted from dry mycelium using a buffer with a high glycerol concentration and thiol protective agent to stabilize enzyme activity. In five steps the enzyme was purified 118-fold. It catalyzed ATP-pyrophosphate exchange in dependence of all three constituent amino acids, and the enzyme could be amino-acylated with L-[14C]valine. The molecular weight of the protein both native (in gel filtration chromatography) and denatured (polyacrylamide gel electrophoresis) was about 220 kDa. These data suggest that delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase consists of a single polypeptide chain and a multienzyme thiotemplate mechanism for the reaction sequence is postulated.     

Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C.

Cephalosporin C was produced by a highly productive strain of Cephalosporium acremonium under industrial production conditions by fed-batch cultivation in a 40-l stirred-tank reactor using a complex medium containing 50 g l-1 peanut flour. The influence of dissolved oxygen concentration (pO2, DOC), which was maintained at different constant levels between 5 and 40% of its saturation value, during the production phase by means of a parameter-adaptive pO2-controller, on the cephalosporin C biosynthesis, was investigated. The concentrations of cephalosporin C (CPC) and its precursors penicillin N (PEN N), deacetoxycephalosporin C (DAOC), and deacetylcephalosporin C (DAC) were monitored by on-line HPLC. The concentrations of amino acids, valine (VAL), cysteine (CYS), alpha-amino-adipic acid (alpha-AAA), the dipeptide alpha-amino-adipyl-cysteine (AC), and the tripeptide alpha-amino-adipyl-cysteinyl-valine (ACV) were determined by off-line HPLC. By reducing the pO2 in the production phase from 40 to 5% of its saturation value, the CPC concentration diminished from 7.2 to 1.1 g l-1 and the PEN N concentration increased from 2.57 to 7.65 g l-1. The DAC concentration also dropped from 3.13 to 0.42 g l-1; however, the DAOC concentration was less influenced. The concentrations of AC and ACV were also less affected. The small DOC did not lead to an accumulation of the intermediate AC and ACV during the production phase. With increasing DOC in the range of 5-20%, the maximal specific production rate, the cell mass concentration-based and the substrate-based yield coefficients for CPC increased almost linearly, and fell back for PEN N.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inactivation of ornithine transcarbamoylase by N delta-(N''-sulpho-diaminophosphinyl)-L-ornithine.

Phaseolotoxin, a tripeptide inhibitor of ornithine transcarbamoylase, is a phytotoxin produced by Pseudomonas syringae pv. phaseolicola, the causal agent of halo-blight in beans. In vivo the toxin is cleaved to release N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine, the major toxic chemical species present in diseased leaf tissue. This paper reports on the interaction between N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine and ornithine transcarbamoylase. N delta-(N'-Sulpho-diaminophosphinyl)-L-ornithine was found to be a potent inactivator of the enzyme, in contrast with phaseolotoxin, which previously has been reported to inhibit the enzyme reversibly. Inactivation by N delta-(N'-[35S]sulpho-diaminophosphinyl)-L-ornithine resulted in the incorporation of 35S into ethanol-precipitated protein. The stoicheiometry of 35S incorporation was approximately 1 mol/mol of active sites. Inactivation was second-order and a rate constant of 10(6) M-1 X s-1 at 0 degree C in 50 mM-Tris/HCl, pH 9.0, was obtained. Carbamoyl phosphate, a substrate of ornithine transcarbamoylase, protected the enzyme from inactivation. A dissociation constant of 3 microM for the enzyme-carbamoyl phosphate complex was calculated. L-Ornithine, the second substrate for ornithine transcarbamoylase, protected the enzyme only at high concentrations. The results are consistent with N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine being a potent affinity label that binds via the carbamoyl phosphate-binding site of ornithine transcarbamoylase. Cleavage of phaseolotoxin to N delta-(N'-sulpho-diaminophosphinyl)-L-ornithine in vivo appears to be an important function in the physiology of the disease.     

Enzymatic synthesis of the penicillin and cephalosporin nuclei from an acyclic peptide containing carboxymethylcysteine

The tripeptide delta-(L- carboxymethylcysteinyl )-L-cysteinyl-D-valine (L-CMC-CV) is converted sequentially into the CMC analog of isopenicillin N, the CMC analog of penicillin N, and the CMC analog of desacetoxycephalosporin C by, respectively, isopenicillin N synthetase, isopenicillin N epimerase, and desacetoxycephalosporin C synthetase, all isolated from the beta-lactam producing prokaryote Streptomyces clavuligerus.