The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa ab heterodimer in an undissociated form Francisco J. Ferna ´ ndez 1 , Rosa E. Cardoza 2 , Eduardo Montenegro 1 , Javier Velasco 1 , Santiago Gutie ´ rrez 1,2 and Juan F. Martı ´ n 1,2 1 Area de Microbiologı ´ a, Facultad de Ciencias Biolo ´ gicas y Ambientales, Universidad de Leo ´ n, Spain; 2 Instituto de Biotecnologı ´ ade Leo ´ n INBIOTEC, Parque Cientı ´ fico de Leo ´ n, Spain The isopenicillin N acyltransferases (IATs) of Aspergillus nidulans and Penicillium chrysogenum differed in their ability to maintain the 40-kDa proacyltransferase ab heterodimer in an undissociated form. The native A. nidulans IAT exhibited a molecular mass of 40 kDa by gel filtration. The P. chrysogenum IAT showed a molecular mass of 29 kDa by gel filtration (corresponding to the b subunit of the enzyme) but the undissociated 40-kDa heterodimer was never observed even in crude extracts. Heterologous expression experiments showed that the chromatographic behaviour of IAT was determined by the source of the penDE gene used in the expression experiments and not by the host itself. When the penDE gene of A. nidulans was expressed in P. chrysogenum npe6andnpe8orinAcremo- nium chrysogenum, the IAT formed had a molecular mass of 40 kDa. On the other hand, when the penDE gene origin- ating from P. chrysogenum was expressed in A. chryso- genum, the active IAT had a molecular mass of 29 kDa. The intronless form of the penDE gene cloned from an A. nidu- lans cDNA library and overexpressed in Escherichia coli formed the enzymatically active 40-kDa proIAT, which was not self-processed as shown by immunoblotting with anti- bodies to IAT. This 40-kDa protein remained unprocessed even when treated with A. nidulans crude extract. In con- trast, the P. chrysogenum penDE intronless gene cloned fromacDNAlibrarywasexpressedinE. coli,andtheIAT was self-processed efficiently into its a (29 kDa) and b (11 kDa) subunits. It is concluded that P. chrysogenum and A. nidulans differ in their ability to self-process their respective proIAT protein and to maintain the a and b subunits as an undissociated heterodimer, probably because of the amino-acid sequence differences in the proIAT which affect the autocatalytic activity. Keywords: enzyme processing; filamentous fungi; gene expression; penicillin biosynthesis. Aspergillus nidulans and Penicillium chrysogenum are able to synthesize hydrophobic penicillins because of substitu- tion of the L -a-aminoadipyl side chain of isopenicillin N by aromatic acyl side chains catalyzed by the isopenicillin N acyltransferase (IAT) [1–3], whereas Acremonium chryso- genum lacks this enzyme [4]. The genes encoding the three enzymes of the penicillin biosynthetic pathway pcbAB [for the multienzyme d(a-aminoadipyl)-cysteinyl-valine synthe- tase], pcbC (for the isopenicillin N synthase) and penDE (for IAT) have been cloned from P. chrysogenum [5–10] and A. nidulans [11–13] and are linked in a cluster [8]. The three genes were found to be very similar in A. nidulans and P. chrysogenum, and the overall organization of the penicillin gene cluster is identical in the two fungi [14,15]. However, wild-type strains of P. chrysogenum areableto synthesize 30-fold higher levels of penicillin than wild-type A. nidulans (their penicillin production levels are about 150 and 5 lgÆmL )1 in shake flasks, respectively) implying that differences in expression of the penicillin biosynthesis genes, or changes in enzyme processing or enzyme activity, may be responsible for the disparity in penicillin biosyn- thetic ability. The last enzyme of the penicillin biosynthetic pathway, IAT, has been shown to be a complex protein which catalyzes five related reactions [16]. The P. chrysogenum IAT is a heterodimer [17,18] composed of two subunits, a (11 kDa, corresponding to the N-terminal region) and b (29 kDa, C-terminal region), which are formed from a 40-kDa precursor protein (proacyltransferase) encoded by the penDE gene [6]. The enzymatic activities and substrate specificity of the proacyltransferase compared with that of the mature enzyme remain obscure. Initial studies on the purification of IAT showed that activity is associated with the 29-kDa subunit [19,20]. Later, Tobin and coworkers [17,18] showed that a higher IAT activity is observed after association of the 29-kDa and 11-kDa subunits, i.e. the most active P. chrysogenum IAT is a heterodimer of these two subunits. Our initial experiments indicated that the chromato- graphic behaviour of A. nidulans IAT differed from that of P. chrysogenum. This prompted us to investigate whether Correspondence to J. F. Martı ´ n, Area de Microbiologı ´ a, Facultad de Ciencias Biolo ´ gicas y Ambientales, Universidad de Leo ´ n, 24071 Leo ´ n, Spain. Fax: + 34 987 291506, E-mail: degjmm@unileon.es Abbreviations: IAT, isopenicillin N acyltransferase; IPTG, isopropyl thio-b- D -galactoside. (Received 31 October 2002, revised 10 December 2002, accepted 10 March 2003) Eur. J. Biochem. 270, 1958–1968 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03561.x there were differences in the post-translational processing of the proIATs of the two fungi that might explain the differences in their penicillin biosynthetic ability. For this purpose, antibodies against the P. chrysogenum and A. nidulans proacyltransferases were prepared. Our study shows that the A. nidulans proIAT remains undissociated as a 40-kDa protein through various purifi- cation steps, whereas the P. chrysogenum enzyme is effi- ciently self-processed, rapidly dissociating into the 29-kDa and 11-kDa subunits. The IAT of A. nidulans purified from Escherichia coli expressing the intronless form of the penDE gene was fully active but was not self-processed. In contrast, the P. chrysogenum IAT obtained in E. coli from an intronless form of its gene was processed into its component subunits. Materials and methods Strains A. nidulans ATCC 28901, a biotin-requiring penicillin- producing strain, was used for biochemical studies and as a source of RNA for constructing the cDNA library. The RNA was obtained from 72-h cultures in MFA medium (containing in gÆL )1 , pharmamedia 25, lactose 70, ammo- nium sulfate 7.5, calcium carbonate 10, biotin 0.02 and potassium phenoxyacetate 6.5, pH 7.2). Similarly, a cDNA library of P. chrysogenum Wis49-408 was obtained from total RNA of 48-h cultures in complex penicillin production CP medium [21]. E. coli XL1-Blue [22], SURE and SOLR (Stratagene, La Jolla, CA, USA) were used for construction of cDNA libraries. E. coli JM109(DE3) [23], a strain that contains a hybrid RNA polymerase gene (lacUV5 promoter coupled to the T7 RNA polymerase gene) integrated into the chromosome (Promega, Madison, WI, USA), was used for isopropyl thiogalactoside-mediated induction of gene expression from the pT7-7 vectors mediated by isopropyl thio-b- D -galactoside (IPTG). The phage kZAP II was used to clone cDNA inserts. These inserts were recovered as either plasmids or phages. The M13 and f1 phage derivatives ExAssist and VCSM13 (Stratagene)wereusedinthein vivo excision of the recombinant cDNA clones. Cell extracts Cell extracts were obtained from 48-h or 72-h cultures of A. nidulans ATCC 28901 or P. chrysogenum AS-P-78 (a high-penicillin-producing strain donated by Antibio ´ ticos S.A., Milan, Italy) in CP medium, which supports efficient penicillin production. Cultures were incubated at 25 °C in a rotary shaker at 250 r.p.m. as described previously [12]. The mycelia were collected by filtration, washed three times with sterile saline solution (9 gÆL )1 NaCl)at4°C, suspended (420 g wet weight) in TD buffer (50 m M Tris/ HClpH8.0,5m M dithiothreitol) and disrupted with glass beads in a mechanical cell disruptor (Braun, Melsungen, Germany) under liquid CO 2 refrigeration. The extract was centrifuged at 20 000 g for 20 min, and the super- natant collected and used for further purification. Self-processing of A. nidulans IAT in cell extracts In experiments to study IAT self-processing ability, extracts of A. nidulans from 72-h cultures containing 7.92 mg proteinÆmL )1 were used. After incubation at different temperatures, 50 lg protein was loaded into each well and immunodetected after electrophoresis with a 1 : 80 000 dilution of antibodies to IAT. IAT assays The activity of A. nidulans or P. chrysogenum IAT was quantified routinely as described previously [16]. One unit is defined as the activity that forms 1 nmol penicillin GÆmin )1 . Specific activities are given as UÆ(mg protein) )1 . The IAT of either P. chrysogenum or A. nidulans was purified after removal of nucleic acids by precipitation with protamine sulfate in 50 m M Tris/HCl, pH 8.0 (final con- centration 0.4%) followed by centrifugation at 20 000 g for 30 min. The proteins in the supernatant were fractionated by precipitation with ammonium sulfate. Most IAT activity was found in the 40–55% ammonium sulfate fraction. Determination of molecular mass ThemolecularmassoftheIATswasestablishedbygel filtration of cell-free extracts on a Sephadex G75 Superfine column (2.6 · 70 cm) calibrated with a set of molecular mass markers (BSA, 67 kDa; ovalbumin, 43 kDa; chymo- trypsinogen, 25 kDa, and ribonuclease A, 13.7 kDa). SDS/PAGE SDS/PAGE was performed as described by Laemmli [24]. Phosphorylase B (94 kDa), BSA (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa) (Pharmacia low molecular mass calibration kit) were used as markers. Expression in E. coli and refolding of IAT Transformants of E. coli JM109(DE3) with the appropriate cDNA constructions of the penDE genes of P. chrysogenum or A. nidulans (see Results) were grown in Luria–Bertani broth/chloramphenicol at 37 °CuntilanA 600 of 0.4 was reached. Expression of the penDE gene was induced by the addition of IPTG (final concentration 0.5 m M ), and after 4 h the induced E. coli cells were harvested by centrifuga- tion. Then 0.5 g E. coli cells were suspended in 10 mL 25 m M Tris buffer containing 5 m M EDTA,pH5.0,and lysed by incubation with lysozyme (final concentration 1mgÆmL )1 )for15minat4°C followed by sonication. The insoluble inclusion bodies were collected by centrifugation at 12 000 g for 10 min and solubilized in 8 M urea in redox buffer (containing 10 m M glutathione, 1 m M oxidised glutathione, 50 m M Tris, 10 m M CaCl 2 and 10 m M MgCl 2 ) at pH 8.0. Proteins were refolded from solubilized inclusion bodies by diluting the redox buffer to a final concentration of 4 M urea and 100 lgproteinÆmL )1 , and supplementing it with poly(ethylene glycol) 6000 [at a final molar ratio of poly(ethylene glycol) to protein of 10] [25]. Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1959 Preparation of antibodies against the P. chrysogenum or A. nidulans IATs After expression of the corresponding genes in pT7-7 vectors [26] the purified inclusion bodies were solubilized and the IAT was purified by semipreparative SDS/PAGE. New Zealand rabbits were immunized by intradermal injection with the pure protein, using the protocol described by Dunbar & Schwoebel [27]. This immunization process was repeated once a month for 3 months using incomplete Freund’s adjuvant. After the immunization was completed, blood serum was collected by centrifugation, and the IgG fraction purified by ammonium sulphate precipitation and FPLC using a Protein A–Sepharose column (Pharmacia Biotech Inc., Uppsala, Sweden) as described by Harlow & Lane [28]. Results Molecular mass of the A. nidulans and P. chrysogenum IAT Gel-filtration chomatography on Sephadex G75 superfine of the A. nidulans IAT revealed a K av of 0.171, which corresponded to a molecular mass of 39 811 Da, suggesting that it is not dissociated into its component subunits. In contrast, the P. chrysogenum IAT was eluted from the same column with a K av of 0.275, which corresponded to a molecular mass of 29 227 Da (Fig. 1A). Processing and dissociation of the protein is determined by the cloned gene and not by the host itself in A. nidulans also. To establish if the lack of processing and dissociation of the A. nidulans IAT was due to intrinsic characteristics of the protein itself or to the absence of a processing endopeptidase in the host, the penDE gene of either P. chrysogenum or A. nidulans was introduced with its own promoter and regulatory sequences into strains npe6 and npe8ofP. chrysogenum (deficient in IAT activity [29,30]) and into A. chrysogenum CW19, a fungus that lacks IAT [4]. Transformants with constructions containing either the P. chrysogenum or A. nidulans penDE gene were selected, and the molecular mass of the IAT was studied by gel filtration through Sephadex G75. All transformants Fig. 1. Comparative molecular mass of P. chrysogenum and A. nidulans IATs when the corresponding genes are expressed in different fungal hosts. (A) K av values deduced by gel filtration on Sephadex G75 superfine of the IATs of A. nidulans and P. chrysogenum,andseveral transformants with the penDE gene. Strain designation AN28901 corresponds to A. nidulans 28901; W54-1255 is P. chrysogenum Wis54- 1255; CW19.3, 8p-1.3 and 6p-1.13 correspond to transformants of A. chrysogenum CW19, P. chrysogenum npe8andP. chrysogenum npe6, respectively, with the penDE gene of P. chrysogenum (molecular mass 29 kDa). Strains CW19.10, 8B1 and 6A2 correspond, respect- ively, to the IATs of A. chrysogenum CW19, P. chrysogenum npe8and P. chrysogenum npe6 transformants with the penDE gene of A. nidu- lans (molecular mass 40 kDa). Ribonuclease A (RA), chymotryp- sinogen A (ChT), ovalbumin (OA), and BSA were used as standards. All transformants were obtained with the corresponding native penDE gene from P. chrysogenum or A. nidulans with their own promoters. (B) Scheme summarizing the host strains and transformants used and the molecular mass of their respective IATs. Dark boxes indicate transformants with the penDE gene of A. nidulans and grey shading corresponds to transformants with the P. chrysogenum penDE gene. 1960 F. J. Ferna ´ ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003 contained one to three integrated intact copies of either the P. chrysogenum or A. nidulans penDE genes as shown by Southern blot hybridization. As shown in Fig. 1B, all A. chrysogenum and P. chrysogenum transformants with the A. nidulans penDE gene formed IAT of molecular mass 39–40 kDa, whereas transformants carrying the P. chryso- genum gene formed an IAT with a molecular mass of 28–29 kDa. These results indicate that the A. nidulans penDE gene, when expressed in P. chrysogenum or A. chrysogenum, forms a protein that is not dissociated into its component subunits, unlike the IAT of P. chrysogenum introduced into the same host strains. Constructions for expressing the A. nidulans IAT a and b subunits and the ab proacyltransferase in E. coli As the 40-kDa proacyltransferase of P. chrysogenum cannot be isolated as the undissociated protein because it is rapidly self-processed in the cell, emphasis was put on the compar- ative study of the activities of the A. nidulans 40-kDa acyltransferase with those of its separate 29-kDa and 11-kDa subunits obtained by expressing the corresponding DNA fragments in E. coli. To prepare constructions for expression in E. coli, six different phages containing cDNA for the A. nidulans penDE gene were isolated from about 30 000 phage plaques. The inserts in the five phages were sequenced. None contained any of the three introns of the penDE gene. Two of the cloned cDNA fragments (pFCaat107 and pFCaat108) showed 5¢ ends that started 86 and 73 bp upstream, respectively, from the initial ATG of the penDE ORF; two others (pFCaat104 and pFCaat106) started 34 or 19 bp, respectively, downstream from the ATG. The fifth phage (pFCaat100) contained an insert that started 315 bp downstream from the ATG, close to the nucleotide position 309–315, which encodes the CTT motif corresponding to the processing site of the proacyl- transferase into its subunits. The 3¢ termini of all cloned cDNA inserts (except pFCaat108) extended past the PstI site located 40 bp downstream from the TGA stop codon of the penDE gene. All inserts were recovered from the phagemids as EcoRI– PstIfragments.TheEcoRI–PstI fragments were subcloned into the pT7-7 vector for overexpression in E. coli.As shown in Fig. 2, a translational fusion expression system was constructed by subcloning the intronless form of penDE gene as an SspI–PstI fragment from pFCaat107 into the expression vector pT7.7. The resulting plasmid carried the entire penDE gene (encoding the a and b subunits) in-frame with a 13 amino-acid fragment of the pT7-7 system. To avoid problems in the assay of IAT due to degradation of isopenicillin N by the penicillinase encoded by the ampicillin resistance gene of the pT7-7 system, the pT7-7-penDE cassette was subcloned as a SmaI–NcoIfragmentinto plasmid pBC KS+ (Stratagene) which contains the chlo- ramphenicol (instead of ampicillin) resistance gene as selective marker, thus obtaining plasmid pULCTaß. Three other plasmids were constructed for overexpression in the pT7-7 system: the a subunit (pULCTa), the b subunit (pULCTb)andthea and b subunits in the same plasmid but on separate ORFs (pULCTa+b). The different constructions are shown in Fig. 2. Two other similar constructions, pULCT106aß (carrying both the a and b subunits as a single ORF, i.e. as they occur in the proacyltransferase) and pULCT100b (with only the b subunit), were constructed from the original inserts in phage pFCaat106 and pFCaat100, respectively, by following similar strategies. All constructions with the A. nidulans intronless form of the penDE gene resulted in good translation of the respective proteins (see below). The 40-kDa A. nidulans proacyltransferase is not processed When the above constructions were introduced into E. coli JM109(DE3), a strain containing the integrated T7 RNA polymerase gene induced by IPTG, analysis of the proteins by PAGE showed that a strong band of the A. nidulans 40-kDa protein is induced in E. coli transformed with pULCTab and pULCT106ab, as expected. About 20% of the total E. coli proteinwasestimatedtobeIATinextracts of E. coli expressing these constructions (Fig. 3A, lanes 4and7). Expression of the 40-kDa protein was also evidenced by autoradiography of the labelled proteins after addition of a pulse of [ 35 S]methionine. Only two proteins, an unknown 25-kDa polypeptide (perhaps a fragment of the T7 RNA polymerase induced by IPTG) and the 40-kDa IAT (Fig. 3B, lanes 4 and 7), were labelled with methionine under these expression conditions. No self-processing of the 40-kDa A. nidulans proIAT was observed in E. coli extracts. The IAT protein of E. coli [pULCTab] always moved as a 40-kDa protein on SDS/ PAGE and no 29-kDa or 11-kDa subunits were observed on either SDS/PAGE or autoradiography of the labelled protein (a highly sensitive method). This result indicates that the A. nidulans proIAT formed in E. coli is not self- processed. Similarly, the 29-kDa (b) subunit was overproduced in E. coli transformedwithpULCTb after 1 and 3 min of induction by IPTG (Fig. 3A, lanes 10 and 11). This protein was clearly separated from the unknown 25-kDa polypep- tide as shown in labelling experiments (Fig. 3B, lane 11). Finally, the 11-kDa (a) subunit was also formed in E. coli transformedwithpULCTa as shown by SDS/PAGE (Fig. 3A, lanes 13 and 14) and by the labelling experiments (Fig. 3B, lane 14). Western blot analysis using the antibodies raised against the A. nidulans 40-kDa proIAT confirmed these results (Fig. 4). As shown in Fig. 4C, the 40-kDa proIAT is formed at either 28 °Cor37 °Cbutnoa or b subunits were detected in the immunoassays. The A. nidulans 40-kDa IAT obtained from E. coli shows acyltransferase activity The E. coli [pULCTab] extracts containing unprocessed A. nidulans proIAT showed acyltransferase activity (Fig. 3C) [102 pmolÆmin )1 Æ(mg protein) )1 ] equivalent to that of the crude extracts of A. nidulans. The reaction product was sensitive to b-lactamase and was identified as penicillin G by HPLC. The b subunit overexpressed in E. coli [pULCTb]did not show IAT activity nor did the a subunit expressed Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1961 separately in E. coli [pULCTa]. No IAT activity was observed when the extracts containing a and b subunits were mixed together or when they were expressed from plasmid pULCTa+b in which the genes encoding both polypeptides are located (Fig. 3C). These results indicate that in A. nidulans the ab 40-kDaIATistheactiveform, whereas the mixture of a and b subunits expressed in E. coli do not reconstitute to an active IAT. A processed biologically active P. chrysogenum IAT is recovered after expression in E. coli Similarly, recombinant phages carrying the P. chrysoge- num intronless form of penDE gene were selected from the cDNA library of P. chrysogenum by hybridization with a 1.6-kb XhoI–XbaI probe containing the penDE gene. The absence of introns was confirmed by sequencing clones containing fragments covering the intron sites; the 5¢ end of the cDNA fragment was confirmed to be 37 nucleotides upstream of the ATG translation initiation codon. Two constructions, pPT7ab and pPBCab (Fig. 5), were assem- bled in which the ORF of the acyltransferase was excised with XmnI endonuclease at the AATG site (coinciding with the ATG translation initiation codon) and linked to the pT7-7 E. coli expression plasmid digested with NdeI, filledwithdTTPandtreatedwithMung-beanexonuclease to remove the protruding nucleotide. After the ligation, the CATGCTT sequence at the linkage site was confirmed by sequencing through the fusion point. In pPBCab the chloramphenicol resistance gene was used as a marker instead of the ampicillin resistance gene present in pPT7ab. As the unprocessed 40-kDa IAT was never recovered from P. chrysogenum extracts, overexpression of the P. chrysogenum penDE gene in E. coli was carried out using constructions pPBCab and pPT7ab. As shown in Fig. 2. Plasmids used to express the a and b subunits of the A. nidulans IAT in E. coli. Restriction endonuclease map of pULCTab,pULCTa, pULCTb and pULCTa+b containing, respectively, the complete penDE gene of A. nidulans, the DNA fragments encoding the a or b subunits, or the DNA regions corresponding to the a and b subunits on different fragments in the same plasmid. In all constructions the DNA fragments encoding the penDE gene (or their fragments) were expressed from phage T7 promoter. Cm R , chloramphenicol resistance gene; Ap R , ampicillin resistance gene; + F indicates filled ends after digestion with restriction endonucleases. 1962 F. J. Ferna ´ ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003 Fig. 6, abundant expression of the 40-kDa ab protein was obtained at 37 °CinE. coli transformed with each of the constructions under induction conditions but not in noninduced conditions (Fig. 6A). The P. chrysogenum 40-kDa IAT could be easily recov- ered as inclusion bodies in large amounts. The enzyme purified in this manner showed no traces of the 29-kDa or 11-kDa polypeptides. However, when the pure 40-kDa P. chrysogenum proIAT was solubilized and refolded by dilution in a redox buffer containing poly(ethylene glycol) and incubated for 6 h at room temperature, these polypeptides were formed by autocatalytic cleavage (Fig. 6C), and the heterodimeric form thus obtained (containing the two subunits) showed IAT activity. Similar results were reported by Tobin et al. [17]. To confirm that there were differences in proIAT processing ability in the two fungi, constructions in E. coli with either the P. chrysogenum or A. nidulans penDE genes were expressed in parallel using [ 35 S]methionine as marker. The results (Fig. 7) clearly indicate that, whereas the 40-kDa protein is rapidly processed in P. chrysogenum when incubated at 28 °C (but not at 37 °C), the A. nidulans remains as a 40-kDa protein when incubated at either 28 °Cor37°C. The self-processing of the P. chrysogenum IAT was confirmed by immunoblot studies using specific antibodies to the P. chrysogenum IAT (Fig. 4). Western blot analysis revealed that the P. chrysogenum 40-kDa proIAT is efficiently self-processed when the gene is expressed at 28 °C but not at 37 °C, giving similar amounts of the 29-kDa and 11-kDa proteins (Fig. 4B, lane 2). Extracts of A. nidulans do not process the 40-kDa IAT obtained in E. coli To exclude the possibility that an A. nidulans peptidase activity was required for in vivo processing, the labelled 40-kDa IAT obtained after expressing the A. nidulans penDE gene in E. coli was incubated for 0, 30 and 60 min with cell-free extracts of A. nidulans (7.92 mg proteinÆmL )1 ) obtained from cells grown in penicillin production condi- tions. The results showed that there is no processing of the protein even after incubation for 60 min. The amount of labelled protein remaining after treatment with the A. nidu- lans extract was approximately the same as that observed with boiled A. nidulans extracts. Fig. 3. Proteins formed after expression of the A. nidu lans IAT a and b subunits and assay of their catalytic activity. SDS/PAGE (A) and autoradiography (B) of proteins expressed in E. coli from different constructions with the penDE gene of A. nidulans.LaneM,molecular massmarkers;lanes1,2,3,controlE. coli [pBC] without insert; lanes 4, 5, 6, E. coli [pULCTab];lanes7,8,9,E. coli [pULCT106ab]; lanes 10, 11, 12, E. coli [pULCTa]; lanes 13, 14, 15, E. coli [pULCTb]. Lanes1,4,7,10and13wereinducedwithIPTG.Lanes2,5,8,11and 14 were induced with IPTG and supplemented with a 15-min pulse of [ 35 S]methionine/[ 35 S]cysteine. Lanes 3, 6, 9, 12 and 15 were not induced. In the autoradiography, note the formation of a 40-kDa labelled protein in lanes 5 and 8 (containing the ab constructions) and proteins of 29 kDa in lane 11, and 11 kDa in lane 4 (containing, respectively, the a or b subunits). A band of about 25 kDa observed in all labelled preparations (lanes 2, 5, 8, 11 and 14) is an unknown protein induced by IPTG. (C) Bioassay of the IAT activity using extracts of E. coli transformed with different constructions with the A. nidulans penDE gene. Formation of benzylpenicillin in the IAT reaction was determined using Bacillus subtilis as test organism. 1, E. coli JM109(DE3) [pULCTab] undiluted extract; 2, E. coli [pUL- CTab]extractdiluted1:2;3,E. coli [pULCTb]; 4, E. coli [pULCTa]; 5, control benzylpenicillin solution (1 lgÆmL )1 ); 6, same as 1 after treatment of the extract with b-lactamase (Bactopenase; Difco); 7, mixture of extracts of E. coli [pULCTa]andE. coli [pULCTb]; 8, E. coli [pULCTa+b]. Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1963 Immunoblot analysis of extracts of P. chrysogenum and A. nidulans shows different in vivo processing of the IAT The availability of antibodies to IAT of P. chrysogenum and A. nidulans allowed us to follow the in vivo processing of IAT in both fungi. As shown in Fig. 8, Western blot analysis of IAT in extracts of cells grown for 48, 72 or 96 h revealed that P. chrysogenum IAT was already completely processed to the 29-kDa (a) and 11-kDa (b) subunit at 48 h and remained processed thereafter; the intensity of the bands increased at 72 and 96 h, in agreement with the enzyme being involved in secondary metabolism. In contrast, the 40-kDa (unprocessed) IAT form (ab)was clearly observed in A. nidulans cultures at 48, 72 and 96 h and the intensity increased at 96 h. Degraded forms were observed in the A. nidulans Western blot, but the 11-kDa band was never observed, indicating that A. nidulans IAT is processed or degraded differently from the P. chrysogenum enzyme. Discussion Formation of mature enzymes from preproenzymes is a common phenomenon in eukaryotic organisms. In most cases, specific endopeptidases are involved in recognition and cleavage of the proenzymes. Some proteins with pepti- dase activity may process themselves autocatalytically [31]. The 40-kDa P. chrysogenum IAT is a heterodimer of a and b subunits [17,18,32]. An important difference between the IATs of P. chrysogenum and A. nidulans is that during purification of the active form of the P. chrysogenum enzyme, the 40-kDa heterodimer is never observed, and instead the b-subunit (29 kDa) is enriched throughout the purification process [20]. The significant loss of total enzyme activity during purification of P. chrysogenum IAT is con- sistent with the fact that only the 29-kDa protein is enriched whereas both subunits are required for full enzyme activity. When the penDE gene of A. nidulans was expressed in IAT-deficient mutants of P. chrysogenum or in A. chryso- genum, the molecular mass of the IAT formed was that of Fig. 5. Restriction map of plasmids pPT7ab and pPBCab containing the pe nDE gene of P. chryso genu m under the T7 promoter. Plasmid pPT7ab contains the ampicillin resistance marker whereas pPBCab contains the chloramphenicol resistance gene. Abbreviations are the same as in Fig. 3. Fig. 4. Comparative expression of the genes pe nDE from P. chrysogenum and A. nidulans in E. coli at two different temperatures and immunodetection of the IAT proteins. (A) SDS/PAGE of the cell lysates. (B) Immunological detection of the IAT protein from P. chrysogenum.(C)Immunological detection of the IAT protein from A. nidulans. Lanes 1, 2, 8 and 9, E. coli [pPBCab], contains the gene penDE from P. chrysogenum (lanes 1 and 8 without IPTG induction and lanes 2 and 9 with 0.5 m M IPTG). Lanes 3, 4, 10 and 11, E. coli [pULCTab], contains the gene penDE from A. nidulans (lanes 3 and 10 without IPTG induction and lanes 4 and 11 with 0.5 m M IPTG). Lane 5, molecular mass markers. Lanes 6 and 7, E. coli [pT7.7] used as control (lane 6 without IPTG induction and lane 7 with 0.5 m M IPTG). Lanes 1–4 and 6–7 contain cell lysates obtained from bacterial cultures grown at 28 °C, and lanes 8–11 contain cell lysates obtained from bacterial cultures grown at 37 °C. 1964 F. J. Ferna ´ ndez et al.(Eur. J. Biochem. 270) Ó FEBS 2003 the A. nidulans enzyme (40 kDa). All available information supports the conclusion that self-processing is determined by the amino-acid sequence of the IAT itself and not by the host. Immunoblot studies using antibodies against P. chryso- genum or A. nidulans IAT supported this conclusion. The P. chrysogenum IAT was already fully processed after 48 h of incubation under penicillin production conditions, whereas the A. nidulans enzyme remained in the 40-kDa form for at least 96 h of incubation. No peptidases able to cleave IAT were found in the fungal extracts. This indicates that processing of P. chrysogenum IAT is autocatalytic and is consistent with the observation of processed enzyme in the hetero- logous Acremonium system when the P. chrysogenum Fig. 6. Processing of the soluble form, inclusion bodies, and refolded forms of the IAT of P. chrysogenum. (A)SDS/PAGEofproteinsexpressedin E. coli [pPBCab]andE. coli [pPT7ab] (containing the penDE gene of P. chrysogenum). Lane 1, noninduced E. coli [pPBCab]; lane 2, induced E. coli [pPBCab]; lane 3, noninduced E. coli [pPT7ab]; lane 4, induced E. coli [pPT7ab]. Note the formation of the 40-kDa protein (arrow). (B) SDS/PAGE of proteins collected as insoluble material (inclusion bodies) after overexpression in E. coli. Lane 1, molecular mass markers; lane 2, total extract of E. coli [pPBCab]; lane 3, supernatant of E. coli [pBCab] extracts after centrifugation at 12 000 g; lane 4, insoluble material collected from extracts of E. coli [pPBCab]; lane 5, total extract of E. coli [pPT7ab]; lane 6, supernatant of E. coli [pPT7ab]; lane 7, insoluble material of E. coli [pPT7ab]. Note the presence in crude extracts and in the insoluble material of the 40-kDa protein (arrow). (C) Lane 1, proteins in the inclusion bodies isolated from E. coli [pPBCab]; lane 2, inclusion bodies of E. coli [pPT7ab]; lane 3, size markers; lane 4, refolded proteins in the inclusion bodies of E. coli [pPBCab]; lane 5, refolded proteins in the inclusion bodies of E. coli [pPT7ab]. Note that after refolding there is partial processing of the 40-kDa protein into subunits a (11 kDa) and b (29 kDa) (arrows). The refolded proteins showed considerable IAT activity, which was not detectable in the insoluble inclusion bodies. Fig. 7. Comparative expression and processing in E. coli of the IATs encoded by the penDE genes of P. chrysogenum and A. nidulans at two different temperatures. (A) SDS/PAGE of proteins. (B) Autoradiography of the gel. Lanes 1 and 2, E. coli [pPBCab]containingthepenDE gene of P. chrysogenum without and with induction (note formation of the 29-kDa and 11-kDa subunits in lane 2); lanes 3 and 4, E. coli [pULCTab] containing the penDE gene of A. nidulans without and with induction (note the formation of the 40-kDa protein and the lack of processing to the 29-kDa and 11-kDa subunits) in lane 4; lane 5, molecular mass markers; lanes 6 and 7, control E. coli [pT7-7] without and with induction; lanes 8 and 9, E. coli [pPBCab] without and with induction; lanes 10 and 11, E. coli [pULCTab] without and with induction. In lanes 1–7, cultures were incubated at 28 °C, and in lanes 8–12 at 37 °C. Ó FEBS 2003 Processing of the isopenicillin N acyltransferase (Eur. J. Biochem. 270) 1965 penDE gene was introduced into this fungus which lacks IAT [4]. IAT of P. chrysogenum and A. nidulans catalyses the third step of penicillin biosynthesis, namely the hydrolysis of the peptide (amide) bond between a-aminoadipic acid and cysteine of the penicillin nucleus (condensed cysteinyl- valine) [33]. In this respect, IAT resembles cysteine pepti- dases, and, indeed, the cleavage site of both P. chrysogenum and A. nidulans IATs is the bond Gly102–Cys103 estab- lished by the amino-acid sequence of the N-terminus of the 29-kDa (b) subunit [6,32,33]. The IAT of P. chrysogenum is strongly inhibited by phenylmethanesulfonyl fluoride [16], a well-known inhibitor of serine proteases and acyltransferases. We proposed previously that the GXS309XG motif is involved in cleavage of phenylacetyl-CoA and binding of the phenyl- acetyl moiety to the enzyme. Indeed, Tobin et al.[34] reported that mutation of Ser309 to Ala abolished IAT activity without affecting cleavage of the enzyme. These authors have also shown that mutation of Ser227 alters cleavage of the enzyme [17]. OurresultsindicatethatP. chrysogenum and A. nidulans differ in their ability to self-process the IAT into a and b subunits. This difference is unlikely to be due to amino-acid sequences around the cleavage site because the sequence 99ARDG*CTT(V/A)YC, which includes the cleavage site (indicated by an asterisk), is conserved in both fungi, although the Val106 to Ala106 substitution in the A. nidulans enzyme may have some effect on cleavage. Similarly, Ser227 is conserved in both fungi, which indicates that the inefficient processing and dissociation in A. nidulans is not due to alteration of this particular residue (which may, rather, be involved in isopenicillin N binding because it is also conserved in several cephalosporin and cephamycin biosynthetic enzymes [34]). However, the two IATs differ in 23.5% of their component amino acids (the two cysteines included), which may explain their different autocatalytic activities. The lack of processing of the A. nidulans 40-kDa IAT when expressed in soluble form in E. coli suggests that the self-processing ability of IAT of A. nidulans is weak compared with that of the P. chrysogenum enzyme. Twenty additional amino acids were present in the E. coli-expressed A. nidulans IAT. Although these amino acids may affect the self-cleaving ability, they were not present in the construc- tion used to transform the filamentous fungi (either P. chrysogenum or A. chrysogenum), and the IATs formed in the fungi remain undissociated, as occurs with the enzyme formed in E. coli.The40-kDaA. nidulans IAT is enzymati- cally active, whereas we did not observe any activity with mixtures of the a and b subunits. Penicillin acylases (amidases) occur in many micro- organisms (reviewed in [35,36]). One of the best known, the E. coli penicillin acylase, consists of two dissimilar subunits derived from a membrane-bound single polypep- tide precursor (proacylase) by autocatalytic processing [37,38]. Autocatalytic processing usually leads to more active forms, and this may explain the observed difference in activity of the P. chrysogenum and A. nidulans IATs. The similarity between the E. coli and fungal penicillin amidases from this mechanistic point of view deserves further studies. P. chrysogenum and A. nidulans differ dramatically in their ability to synthesize penicillin, although the gene clusters are similar and occur in a single copy in the wild- type strains of both fungi. Crude extracts of wild-type P. chrysogenum strains show about fivefold higher IAT activity than wild-type A. nidu- lans strains (E. Montenegro and J.F. Martı ´ n, unpublished results). It is possible that the difference in the ability to self- process their respective IATs may affect the overall penicillin-biosynthetic ability of the two fungi. Fig. 8. Western blot analysis showing the different processing of the IATs of P. chrysogenum and A. nidulans. Extracts of P. chrysogenum AS-P-78 and A. nidulans ATCC 28901 grown in CP medium and MFA medium, respectively for 48, 72 or 96 h were obtained, resolved by SDS/PAGE, and visualized with antibodies as described in Materials and methods. Lanes: M, molecular mass markers; 48, 48-h extracts; 72, 72-h extracts; 96, 96-h extracts. The size of the molecular mass markers in kDa is shown on the left. The immunoreactive IAT bands of 40 kDa, 29 kDa and 11 kDa are indicated by arrows on the right. Note the presence of the 40-kDa band and the absence of the 11-kDa IAT band in the A. nidulans extracts. 1966 F. J. Ferna ´ ndez et al.(Eur. J. 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The isopenicillin N acyltransferases of Aspergillus nidulans and Penicillium chrysogenum differ in their ability to maintain the 40-kDa ab heterodimer in an undissociated form Francisco. of the 29-kDa and 11-kDa subunits in lane 2); lanes 3 and 4, E. coli [pULCTab] containing the penDE gene of A. nidulans without and with induction (note the formation of the 40-kDa protein and