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.     

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum.

The content of alpha-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into alpha-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of alpha-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing alpha-aminoadipic acid accumulation. The repression of alpha-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of alpha-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts alpha-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.     

Regulation and compartmentalization of β‐lactam biosynthesis

Penicillins and cephalosporins are β-lactam antibiotics widely used in human medicine. The biosynthesis of these compounds starts by the condensation of the amino acids l -α-aminoadipic acid, l -cysteine and l -valine to form the tripeptide δ-l -α-aminoadipyl-l -cysteinyl-d -valine catalysed by the non-ribosomal peptide ‘ACV synthetase’. Subsequently, this tripeptide is cyclized to isopenicillin N that in Penicillium is converted to hydrophobic penicillins, e.g. benzylpenicillin. In Acremonium and in streptomycetes, isopenicillin N is later isomerized to penicillin N and finally converted to cephalosporin. Expression of genes of the penicillin (pcbAB, pcbC, pendDE) and cephalosporin clusters (pcbAB, pcbC, cefD1, cefD2, cefEF, cefG) is controlled by pleitropic regulators including LaeA, a methylase involved in heterochromatin rearrangement. The enzymes catalysing the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) are located in microbodies, as shown by immunoelectron microscopy and microbodies proteome analyses. Similarly, the Acremonium two-component CefD1–CefD2 epimerization system is also located in microbodies. This compartmentalization implies intracellular transport of isopenicillin N (in the penicillin pathway) or isopenicillin N and penicillin N in the cephalosporin route. Two transporters of the MFS family cefT and cefM are involved in transport of intermediates and/or secretion of cephalosporins. However, there is no known transporter of benzylpenicillin despite its large production in industrial strains.     

Specificity of Penicillin Acylase of Fusarium and of Penicillium chrysogenum

Extracts containing penicillin acylase were obtained by shaking the mycelium of Fusarium avenaceum and of Penicillium chrysogenum in 0.2 M sodium acetate or sodium chloride solution. The optimum pH for conversion of penicillin V into 6-aminopenicillanic acid (6-APA) by the enzyme of Fusarium was about 7.5, and the reaction velocity was increased by a rise in temperature from 27 to 37 C. Penicillin G and penicillins with an aliphatic side chain were cleaved much less readily than was penicillin V. With the enzyme preparation obtained from a nonpenicillin-producing strain of P. chrysogenum, the reaction rate was higher at pH 8.5 than at pH 7.5 and pH 6.5. The acylase of P. chrysogenum hydrolyzes penicillin V more readily than penicillin G. In a series of aliphatic penicillins, the amount of 6-APA formed through the action of this enzyme increased with the number of carbon atoms of the side chain. Penicillins with a glutaryl or an adipyl group as side chain were unaffected by the enzyme of Fusarium and of Penicillium. No reaction was observed upon incubation of penicillin N (with a D-aminoadipyl side chain) or isopenicillin N (with an L-aminoadipyl side chain) with Fusarium and Penicillium extract. When the carboxy group of the side chain of these penicillins was esterified, formation of 6-APA was observed upon incubation with Penicillium extract, whereas no 6-APA or only very small amounts were obtained by acylase of Fusarium.     

New insights into the isopenicillin N transport in Penicillium chrysogenum

In Penicillium chrysogenum the beta-lactam biosynthetic pathway is compartmentalized. This fact forces the occurrence of transport processes of penicillin-intermediate molecules across cell membranes. Many aspects around this molecular traffic remain obscure but are supposed to involve transmembrane transporter proteins. In the present work, an in-depth study has been developed on a Major Facilitator-type secondary transporter from P. chrysogenum named as PenM. The reduction of penM expression level reached by penM targeted silencing, leads to a decrease in benzylpenicillin production in silenced transformants, especially in SilM-35. On the contrary, the penM overexpression from a high efficiency promoter increases the benzylpenicillin production and the expression of the biosynthetic genes. Moreover, when the silenced strain SilM-35 is cultured under penicillin production conditions with 6-aminopenicillanic acid supplementation, an increase in the benzylpenicillin production proportional to the 6-aminopenicillanic acid availability is observed. By this phenomenon, it can be concluded that due to the penM silencing the benzylpenicillin transport remains intact but the peroxisomal isopenicillin N import results affected. As a culminating result, obtained by the expression of the fluorescent recombinant PenM-DsRed protein, it was determined that PenM is naturally located in P. chrysogenum peroxisomes. In summary, our experimental results suggest that PenM is involved in penicillin production most likely through the translocation of isopenicillin N from the cytosol to the peroxisomal lumen across P. chrysogenum peroxisomal membrane.     

Synthesis of tritium-labelled isopenicillin N, penicillin N and 6-aminopenicillanic acid.

1. Phenoxymethylpenicillin sulphoxide 4-methoxybenzyl ester was labelled with 3H in its 2-beta-methyl group. Its specific radioactivity was 362 mCi/mmol. 2. Removal of the side chain of this compound yielded the corresponding ester of 6-aminopenicillanic acid sulphoxide and coupling of the latter with the appropriate protected alpha-aminoadipic acid gave 4-methoxybenzyloxycarbonylisopenicillin N sulphoxide di-4-methoxybenzyl ester or the corresponding derivative of penicillin N. 3. Removal of the protective groups by hydrogenolysis and reduction of the sulphoxide group yielded 3H-labelled isopenicillin N or penicillin N. 4. 3H-labelled phenoxymethylpenicillin sulphoxide was obtained by hydrogenolysis from its 4-methoxybenzyl ester. Reduction of its sulphoxide group and subsequent removal of the side chain gave 3H-labelled 6-aminopenicillanic acid.     

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of δ-(L-α-aminoadipyl)-l-cysteinyl-d-valine and isopenicillin N biosynthesis in Penicillium chrysogenum by the α-aminoadipate pool size

The effect of changes in the intracellular concentration of alpha-aminoadipate on the formation of alpha-aminoadipyl-cysteinyl-valine (ACV) and isopenicillin N (IPN)--two intermediates of penicillin biosynthesis--by strains of Penicillium chrysogenum has been investigated by measuring the incorporation of radioactivity from (6-14C)-alpha-aminoadipate into cellular 14C-ACV and 14C-IPN. No ACV or IPN were found in any strain during cultivation on glucose, but were clearly detected in all three strains during growth on lactose, displaying increased formation in strains exhibiting increased penicillin productivity and increased intracellular alpha-aminoadipate pools. ACV and IPN formation was affected by subjected P. chrysogenum mycelia to either general amino acid control (by addition of amitrol) or by exogenous addition of 5 mM L-lysine. In all cases, the changes observed paralleled the changes in the intracellular alpha-aminoadipate pool. These results are consistent with the alpha-aminoadipate pool limiting the biosynthesis of ACV and IPN and hence penicillin biosynthesis in the present strains of P. chrysogenum.     

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.     

Purification and properties of isopenicillin N epimerase from Streptomyces clavuligerus

Isopenicillin N epimerase, which catalyzes conversion of isopenicillin N to penicillin N, has been purified to electrophoretic homogeneity from the cell-free extract of Streptomyces clavuligerus by a procedure involving ammonium sulfate fractionation and chromatographies with DE-52, DEAE Affi-gel blue, Sephadex G-200, calcium phosphate-cellulose, and Mono Q. The purified epimerase is monomeric with a molecular weight of 47,000 or 50,000 as estimated by SDS-polyacrylamide gel electrophoresis or gel filtration, respectively. The enzyme contains 1 mol of pyridoxal 5'-phosphate per mol of protein, and shows absorption maxima at 280 and 420 nm. The epimerase catalyzes the complete 'racemization' on both the L-alpha-aminoadipyl side-chain of isopenicillin N and the D-alpha-aminoadipyl side-chain of penicillin N, so that an approximately equimolar mixture of the two penicillins is produced. The mixture is not truly racemic, since these penicillins are diastereomers rather than optical isomers. The chemical modification of primary amino groups of the epimerase by fluorescamine results in a great loss of the enzyme activity. The activity of purified enzyme is partially stimulated by the addition of sulfhydryl compounds. The activity is strongly inhibited by sulfhydryl group modifiers such as p-chloromercuribenzoate and N-ethylmaleimide.     

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.     

The transport of phenylacetic acid across the peroxisomal membrane is mediated by the PaaT protein in Penicillium chrysogenum

Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl–CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT–DsRed and EGFP–SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.     

Histamine Acting on H2 Receptors Stimulates Phospholipid Methylation in Synaptic Membranes of Rat Brain

Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[methyl-3H]methionine. The transient increase of [3H]methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 microM) and decreased the basal level in 60 s. Histamine-stimulated [3H]methyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60-65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01-10 microM) or famotidine (0.01-1.0 microM) in a concentration-dependent manner but not by mepyramine (0.1-10 microM) or diphenhydramine (0.1-10 microM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01-10 microM) or dimaprit (1.0 microM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 microM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.     

Cell-free conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine into an antibiotic with the properties of isopenicillin N in Cephalosporium acremonium.

Cell-free extracts of antibiotic-negative mutants of Cephalosporium acremonium converted delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (LLD-tripeptide) into an antibiotic that was destroyed by penicillinase. The enzymic activity of the extracts was destroyed by boiling, but was not inhibited by cycloheximide. LLL-Tripeptide was totally inactive as substrate. The product resembled isopenicillin N, but not penicillin N, in its antibacterial spectrum. We propose that isopenicillin N is the first product of cyclization of LLD-tripeptide.     

Purification and characterization of a 2-oxoglutarate-linked ATP-independent deacetoxycephalosporin C synthase of Streptomyces lactamdurans

The deacetoxycephalosporin C (DAOC) synthase (expandase) of Streptomyces lactamdurans was highly purified, as shown by SDS-PAGE and isoelectric focusing. The enzyme catalysed the oxidative ring expansion that converts penicillin N into DAOC. The enzyme was very unstable but could be partially stabilized in 25 mM-Tris/HCl, pH 9.0, in the presence of DTT (0.1 mM). The enzyme required 2-oxoglutarate, oxygen and Fe2+, but did not need ATP, ascorbic acid, Mg2+ or K+. The optimum temperature was between 25 and 30 degrees C. The DAOC synthase showed a high specificity for the penicillin substrate. Only penicillin N but not isopenicillin N, penicillin G or 6-aminopenicillanic acid served as substrates. 2-Oxoglutarate analogues were not used as substrates although 2-oxobutyrate and 3-oxoadipate inhibited the enzyme by 100% and 56% respectively. The enzyme was strongly inhibited by Cu2+, Co2+ and Zn2+. The apparent Km values for penicillin N, 2-oxoglutarate and Fe2+ were 52 microM, 3 microM and 71 microM respectively. The enzyme was a monomer with a molecular mass of 27,000 Da +/- 1,000.     

Important sites of lipogenesis in the mouse other than liver and white adipose tissue

In a cell-free system prepared by osmotic lysis of protoplasts of Cephalosporium acremonium, isopenicillin N is converted into penicillin N. The epimerase activity of the system is labile.     

Studies on the ring-cyclization and ring-expansion enzymes of beta-lactam biosynthesis in Cephalosporium acremonium

Micrococcus luteus was found to be very sensitive to isopenicillin N and was used as assay organism for purification of the enzyme isopenicillin N synthetase, which cyclizes delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Purification of the enzyme from the crude extract obtained by sonication of mycelia of Cephalosporium acremonium CW-19 was carried out by ammonium sulfate precipitation, desalting with Sephadex G-25, gel filtration on LKB ultrogel AcA44 or ion-exchange chromatography on DEAE-Sepharose. The cyclization enzyme was separated from the ring-expansion enzyme and was purified considerably more than 50-fold by this procedure. Using the purified enzyme, we found that the disulfide bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine required reduction to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine in order to behave as a substrate. The enzyme activity was stimulated by FeSO4 and ascorbate, but other cofactors, including alpha-ketoglutarate, were inactive. In addition to delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, the enzyme converted adipyl-L-cysteinyl-D-valine, N-acetyl-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine, and glycyl-delta-(L-alpha-aminoadipyl)L-cysteinyl-D-valine to penicillins. All of these latter peptides were competitive inhibitors of the cyclization reaction. The Km of the cyclization enzyme is 10 times higher than that of the ring-expansion enzyme, deacetoxycephalosporin C synthetase. The pH and temperature optima of the two enzymes were rather similar. Phosphate inhibited ring expansion, but not cyclization. Both enzymes appear to be soluble enzymes of about 31 000 molecular weight.