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1 Heinrich-Heine-Universität Düsseldorf, Institut für Pharmazeutische Biologie und Biotechnologie, Universitätsstrasse 1, D-40225 Düsseldorf, Germany
2 Eberhard-Karls-Universität Tübingen, Pharmazeutische Biologie, Auf der Morgenstelle 8, D-72076 Tübingen, Germany
Correspondence
Shu-Ming Li
shuming.li{at}uni-duesseldorf.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the nucleotide sequence reported in this paper is EF539173.
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INTRODUCTION |
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), for example: the immunosuppressive cyclosporin A is obtained from Tolypocladium inflatum (Thali, 1995); statins are produced by several fungi, including Aspergillus terreus (Manzoni & Rollini, 2002); penicillins are produced by Penicillium chrysogenum and Aspergillus nidulans (Brakhage et al., 2004); and ergot alkaloids from Claviceps purpurea show diverse pharmacological activities (Schardl et al., 2006). Secondary metabolites from fungi are usually active as mycotoxins and virulence factors (Bennett & Klich, 2003; Rementeria et al., 2005) that could be involved in the pathogenic development of fungal infections. Aspergillus fumigatus, a filamentous fungal saprophyte found ubiquitously in the environment, is responsible for allergenic reactions and invasive aspergillosis, which is a life-threatening disease for immunocompromised patients (Denning et al., 2003). It has been discussed that the virulence of A. fumigatus could be based on its numerous secondary metabolites, especially gliotoxin (Latgé, 1999), which is an epipolythiodioxopiperazine (ETP) derived from Phe and Ser (Gardiner & Howlett, 2005). However, the role of these compounds remains questionable, and needs to be investigated more thoroughly (Latgé, 1999). ETPs are characterized by the presence of an internal disulphide bridge (Gardiner et al., 2005). The role of gliotoxin in the pathogenic development of A. fumigatus has been demonstrated by deleting the non-ribosomal peptide synthetase (NRPS) gene gliP from the gliotoxin biosynthetic gene cluster (Cramer et al., 2006a; Kupfahl et al., 2006), and it was clearly demonstrated that gliotoxin production was blocked in the resulting mutants; however, the abolition of gliotoxin production had no effect on the development of invasive aspergillosis by the mutants (Cramer et al., 2006a; Kupfahl et al., 2006). This proved that gliotoxin is not, or at least not alone, responsible for the pathogenic development in A. fumigatus. Therefore, it would be useful to identify genetic information of further secondary metabolites, which could function as virulence factors.
From the genome sequence of A. fumigatus Af293, at least 26 biosynthetic gene clusters for secondary metabolites have been identified (Nierman et al., 2005), including the gene clusters of fumitremorgin B and fumigaclavine C, as well as that of gliotoxin (Cramer et al., 2006b; Gardiner & Howlett, 2005; Grundmann & Li, 2005; Maiya et al., 2006; Unsöld & Li, 2005). One putative gene cluster has been proposed for the biosynthesis of an additional unknown ETP derivative (Cramer et al., 2006b). This putative gene cluster contains three genes showing significant sequence similarity to gliC, gliP and gliM of the gliotoxin cluster of A. fumigatus (Gardiner & Howlett, 2005). Homologues of these genes, i.e. sirC, sirP and sirM, have also been found in the gene cluster of sirodesmin, which is an ETP derivative from the phytopathogenic fungus Leptosphaeria maculans (Gardiner et al., 2004). The gene cluster of sirodesmin contains a putative prenyltransferase gene, sirD, which has been proposed to be responsible for the O-prenylation of L-Tyr or cyclo-L-Tyr-L-Ser (Gardiner et al., 2004, 2005). A putative prenyltransferase gene, Afu3g12930, has also been found in the cluster from A. fumigatus, and the protein that it encodes shares a sequence identity of 34 % with SirD at the amino acid level; this is higher than the sequence similarity to other fungal prenyltransferases, or other entries in the databases, with the exception of its orthologues in Neosartorya fischeri and A. terreus (see Discussion). Based on this sequence similarity, it could be speculated that EAL92290, encoded by Afu3g12930, would catalyse the transfer reaction of a prenyl moiety to Tyr or its derivatives. To prove the function of Afu3g12930, we cloned and overexpressed this gene in Escherichia coli, and carried out biochemical investigation using the purified fusion protein. Our results showed, interestingly, that Afu3g12930 encodes a second dimethylallyltryptophan synthase (DMATS), termed 7-DMATS, in A. fumigatus, and this enzyme catalyses the prenylation of Trp instead of Tyr. The first DMATS (FgaPT2) identified in A. fumigatus is involved in the biosynthesis of fumigaclavine C; in contrast to this, 7-DMATS catalyses the prenylation of Trp at the C-7 position, instead of at the C-4 position, of the indole ring.
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METHODS |
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Chemicals.
The trisammonium salt of dimethylallyl diphosphate (DMAPP) was synthesized using methods analogous to those used for the synthesis of trisammonium geranyl diphosphate reported by Woodside et al. (1988). Geranyl diphosphate (GPP) was kindly provided by Wessjohann. The peptides used in this study were purchased from Bachem.
Bacterial strains, plasmids and culture conditions.
pGEM-T and pQE60 were obtained from Promega and Qiagen, respectively. A Uni-ZAP XR premade library of A. fumigatus strain B5233 (ATCC 13073) was purchased from Stratagene, and used to obtain phagemids as cDNA templates for PCR amplification. E. coli XL1 Blue MRF' (Stratagene) was used for cloning and expression experiments, and it was grown in liquid Luria–Bertani (LB) medium, or on solid LB medium with 1.5 % (w/v) agar, at 37 °C (Sambrook & Russell, 2001). Addition of 50 µg carbenicillin ml–1 was used for selection of recombinant E. coli strains.
DNA isolation, PCR amplification and cloning.
Standard procedures for DNA isolation and manipulation were performed, as described (Sambrook & Russell, 2001). PCR amplification was carried out on an iCycler from Bio-Rad. Using the Expand High Fidelity kit (Roche Diagnostics), a PCR fragment of 1425 bp, containing the entire coding sequence of Afu3g12930, was amplified from the cDNA library by using the primers 7-dmats-1 (5'-CACCATGGCCATCGGAGCCGAGAT-3') at the 5' end, and 7-dmats-2 (5'-TGCAGATCTGCTGTACACCCGGAG-3') at the 3' end of the gene. Bold letters represent mutations inserted to give the underlined restriction sites NcoI, located at the start codon in 7-dmats-1, and BglII, located at the predicted stop codon in 7-dmats-2. The PCR fragment was cloned into pGEM-T, resulting in plasmid pLW39, which has subsequently had its sequence confirmed (MWG-Biotech). To create the expression vector pLW40, pLW39 was digested with NcoI and BglII, and the resulting NcoI–BglII fragment of 1418 bp was ligated into pQE60, which had been digested with the same enzymes.
Overproduction and purification of His6-7-DMATS protein.
For gene expression, E. coli XL1 Blue MRF' cells harbouring the plasmid pLW40 were cultivated in 300 ml Erlenmeyer flasks containing 100 ml liquid LB medium, supplemented with carbenicillin (50 µg ml–1), and grown at 37 °C to an OD600 of 0.6. For induction, IPTG was added to a final concentration of 0.8 mM, and the cells were cultivated for a further 16 h at 37 °C, before harvest. The bacterial cultures were centrifuged, and the pellets were resuspended in lysis buffer (10 mM imidazole, 50 mM NaH2PO4 and 300 mM NaCl, pH 8.0) at between 2 and 5 ml (g wet weight)–1. After addition of 1 mg lysozyme ml–1, and incubation on ice for 30 min, the cells were sonicated six times, for 10 s each time, at 200 W. To separate the cellular debris from the soluble proteins, the lysate was centrifuged at 14 000 g for 30 min at 4 °C. One-step purification of the recombinant His6-tag fusion protein by affinity chromatography with Ni-NTA agarose resin (Qiagen) was carried out according to the manufacturer's instructions. The protein was eluted with 250 mM imidazole in 50 mM NaH2PO4 and 300 mM NaCl, pH 8.0. In order to change the buffer, the protein fraction was passed through a NAP-5 column (GE Healthcare), which had been equilibrated with 50 mM Tris/HCl and 15 % (v/v) glycerol, pH 7.5. His6-7-DMATS was eluted with the buffer described, and stored at –80 °C for enzyme assays.
Protein analysis.
Proteins were analysed by SDS-PAGE, which was carried out according to the method of Laemmli (1970), and they were stained with Coomassie brilliant blue G-250.
Assays for 7-DMATS activity.
All the enzyme assays contained 50 mM Tris/HCl, pH 7.5, 1.2–3.8 % (v/v) glycerol, and 10 mM CaCl2. They differed from each other by incubation volume and time, substrate concentration, and amount of recombinant 7-DMATS. The reaction mixtures were incubated at 37 °C, and the reactions were terminated by addition of 10 µl TCA (1.5 M) per 100 µl reaction volume. After removal of the protein by centrifugation (14 000 g, 10 min, 4 °C), the enzymic products were analysed on an HPLC system, as described below. The standard assays for determination of the substrate specificity (100 µl) contained 2 mM L-Trp or another aromatic substrate, 1 mM DMAPP and 2.5 µg purified 7-DMATS, and they were incubated for 45 min. Two independent assays were carried out for quantification. The assay for the isolation of the enzymic product for structural elucidation (5 ml) contained 400 µg purified 7-DMATS, and was incubated for 16 h. The reaction mixture was concentrated on a rotation evaporator at 35 °C to a volume of 750 µl, before injection of 100 µl into HPLC for isolation of 7-dimethylallyltryptophan (7-DMAT). The assays for determination of the kinetic parameters of L-Trp contained 1 mM DMAPP, 2.5 µg 7-DMATS, and L-Trp at a final concentration of 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 3.0 or 5.0 mM, in a total volume of 100 µl. The incubation time was 45 min. For determination of the kinetic parameters of DMAPP, 2.5 µg 7-DMATS, 1 mM L-Trp, and DMAPP at a final concentration of 0, 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.5 or 1.0 mM, were used.
HPLC analysis and isolation of 7-DMAT.
Reaction mixtures of 7-DMATS were analysed on an Agilent HPLC Series 1100 by using an Eclipse XBD-C18 column (4.6x150 mm, 5 µm) at a flow rate of 1 ml min–1. Water (solvent A) and acetonitrile (solvent B), each containing 0.5 % (v/v) TFA, were used as solvents. A gradient was run from 15 to 70 % solvent B in 15 min. After washing with 100 % solvent B for 5 min, the column was equilibrated with 85 % solvent A for 5 min. The substances were detected with a Photo Diode Array detector. 7-DMAT was isolated under the same conditions.
Spectroscopic data.
A 1H-NMR spectrum of 7-DMAT was taken on an Avance DRX 500 spectrometer (Bruker) using 0.1 M DCl in D2O as the solvent. The solvent signal at 4.81 p.p.m. was used as reference. 
(p.p.m.): 7.53 (d, 7.6 Hz, H-4), 7.32 (s, H-2), 7.12 (t, 7.9 Hz, H-5), 7.10 (d, 8.2, H-6), 5.46 (br t, 7.0 Hz, H-2'), 4.39 (t, 6.1, H-11), 3.58 (d, 7.0 Hz, 2H-1'), 3.52 (dd, 15.5, 5.4 Hz, H-10), 3.43 (dd, 15.5, 7.0 Hz, H-10), 1.74 (s, 3H-5'), 1.72 (s, 3H-4').
Positive and negative electrospray ionization (ESI) mass spectra were obtained with a ThermoFinnigan TSQ Quantum. The mass spectrometer was coupled with an Agilent HPLC series 1100 equipped with an RP18-column (2x250 mm, 5 µm). For separation, the column was run with 10 % solvent B (CH3OH) in solvent A (H2O) [each containing 0.1 % (v/v) HCOOH] for 5 min, followed by a gradient from 10 to 100 % solvent B over 30 min. After washing with 100 % solvent B for 10 min, the column was equilibrated with 10 % solvent B for 10 min. The flow rate was at 0.2 ml min–1.
MS data of 7-DMAT.
Positive ESI-MS: [M+H]+: m/z 273.1; ms2 of [M+H]+; m/z (%): 272.9 (5), 256.0 (100), 240.8 (9); 214.0 (64), 212.0 (24), 199.9 (25), 156.0 (6). Negative ESI-MS: [M–H]–: m/z 271.2; ms2 of [M–H]– ; m/z (%): 271.2 (100), 227.3 (40), 210.3 (65); 184.3 (94).
MS data of the prenylated H-L-Trp-L-Gly-OH.
Positive ESI-MS: [M+H]+: m/z 330.2; ms2 of [M+H]+; m/z (%): 313.0 (25), 271.1 (34), 257.0 (41), 244.9 (16), 238.0 (47), 227.0 (7), 212.0 (100), 198.1 (25), 156.1 (11). Negative ESI-MS: [M–H]–: m/z 328.4; ms2 of [M–H]– ; m/z (%): 328.2 (100), 284.3 (31), 225.3 (6), 198.2 (10), 131.1 (39).
MS data of the prenylated cyclo-L-Trp-L-Gly.
Positive ESI-MS: [M+H]+: m/z 312.2; ms2 of [M+H]+; m/z (%): 256.0 (9), 198.1 (100). Negative ESI-MS: [M–H]–: m/z 310.6; ms2 of [M–H]–; m/z (%): 310.2 (31), 113.1 (100).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) at the amino acid level, 31 % with FgaPT2 (DMATS) from A. fumigatus (Unsöld & Li, 2005), 28 % with DMATS from C. purpurea and Claviceps fusiformis (Tsai et al., 1995; Tudzynski et al., 1999), and 28 % with FtmPT1 (Grundmann & Li, 2005) and 26 % with FgaPT1 (Unsöld & Li, 2006), which are both from A. fumigatus. SirD is proposed to catalyse the O-prenylation of L-Tyr or cyclo-L-Tyr-L-Ser in the biosynthesis of sirodesmin (Gardiner et al., 2004). DMATS from different sources catalyse the prenylation of L-Trp at the C-4 position of the indole ring in the biosynthesis of ergot alkaloids (Li & Unsöld, 2006; Schardl et al., 2006). FtmPT1 and FgaPT1 have been found to catalyse the prenylation reactions at the C-2 position of the indole rings of brevianamide F and fumigaclavine A (Grundmann & Li, 2005; Unsöld & Li, 2006), respectively. Based on the high sequence similarity to SirD, we speculate that 7-DMATS could be responsible for a transfer reaction of a prenyl moiety to L-Tyr or its derivatives.
Cloning of Afu3g12930, and overproduction and purification of His6-7-DMATS
The coding region of Afu3g12930 was amplified using PCR from cDNA of A. fumigatus strain B5233 (available in the form of phagemids isolated from a cDNA library), and cloned into the cloning vector pGEM-T. For gene expression, the coding sequence of Afu3g12930 was released from pGEM-T, and cloned into the vector pQE60 (see Methods), resulting in the expression plasmid pLW40.
E. coli cells harbouring pLW40 were induced by 0.8 mM IPTG at 37 °C. His6-7-DMATS was purified with Ni-NTA agarose to apparent homogeneity, as judged by SDS-PAGE (Fig. 1), and a protein yield of 5 mg purified His6-tagged 7-DMATS per litre of culture was obtained. The observed molecular mass was 50 kDa, and this corresponded well to the calculated value of 54 kDa for His6-7-DMATS.
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). Dependence of the product formation on the amount of protein was found up to 4 µg per 100 µl assay, and on a reaction time of up to 50 min. Product formation was strictly dependent on the presence of His6-7-DMATS, L-Trp and DMAPP. The enzyme 7-DMATS was found to be specific for DMAPP. Product formation was observed with DMAPP only, and not with GPP. In contrast, 7-DMATS showed relative flexible substrate specificity towards its aromatic substrates. D-Trp was accepted by 7-DMATS, with a relative activity of 15.5 % of its enantiomer L-Trp. Linear and cyclic dipeptides were also substrates of 7-DMATS. Product formation was clearly observed when H-L-Trp-L-Gly-OH or cyclo-L-Trp-L-Gly was used instead of L-Trp in the incubation mixtures (Table 1). However, the linear tripeptide L-Trp-L-Trp-L-Trp was not accepted by 7-DMATS. No product formation was observed in incubation mixtures with L-Phe, or L-Phe-containing cyclic dipeptides (Table 1). These results demonstrated that L-Trp was the best substrate for 7-DMATS, under our test conditions.
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ESI-MS proved that the enzymic product is a prenylated derivative of Trp by detection of [M+H]+ and [M–H]– at m/z 273 and m/z 271, respectively. Comparison of the 1H-NMR spectrum of the isolated compound with that of L-Trp revealed the presence of signals for a dimethylallyl moiety at 5.46 (br t, 7.0 Hz, H-2'), 3.58 (d, 7.0 Hz, 2H-1'), 1.74 (s, 3H-5') and 1.72 p.p.m. (s, 3H-4'). Correspondingly, signals for four protons instead of five protons were found in the region of the aromatic protons of the isolated compound, i.e. signals for three vicinal protons at 7.53 (d, 7.6 Hz), 7.12 (t, 7.9 Hz) and 7.10 p.p.m. (d, 8.2), and a singlet at 7.32 p.p.m. This indicated that the prenylation had taken place at the C-4 or C-7 position of the indole moiety. The spectrum of the isolated compound differed clearly from that of 4-DMAT for all of the protons, with the exception of the proton at C-2 (Unsöld & Li, 2005). 4-DMAT, the first pathway-specific intermediate in the biosynthesis of ergot alkaloids (Schardl et al., 2006; Unsöld & Li, 2005), is also formed from L-Trp by a prenyl-transfer reaction catalysed by DMATS, e.g. FgaPT2 from A. fumigatus (Unsöld & Li, 2005). The enzymic product of 7-DMATS showed a similar, but different, retention time to that of FgaPT2 on the HPLC chromatogram (Fig. 2
). Therefore, the enzymic product of 7-DMATS was identified as 7-DMAT (Fig. 3). The enzymic products of H-L-Gly-L-Trp-OH and cyclo-L-Trp-L-Gly were unequivocally proven as prenylated derivatives by detection of [M+H]+ at m/z 330.2 and 312.2, as well as (M–H)– at m/z 328.4 and 310.6, respectively.
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), LtxC from Lyngbya majuscula (Edwards & Gerwick, 2004), DMATS from C. purpurea (Gebler & Poulter, 1992), and FgaPT1, FgaPT2 and FtmPT1 from A. fumigatus (Grundmann & Li, 2005; Unsöld & Li, 2005, 2006). The 7-DMATS reaction apparently followed Michaelis–Menten kinetics. By using Hanes–Woolf and Lineweaver–Burk plots, the Km values were determined as 137 µM for L-Trp, and 67 µM for DMAPP. The maximum reaction velocity observed with 7-DMATS was 0.21 µmol min–1 mg–1. These values are in the normal range of those of enzymes involved in the biosynthesis of secondary metabolites. Given that L-Trp was shown to be the best substrate in our test conditions (see above), it is very likely to be the natural substrate of 7-DMATS.
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DISCUSSION |
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), from the same fungus; this level of identity is slightly higher than the sequence similarities between 7-DMATS and other indole prenyltransferases from A. fumigatus, e.g. 7-DMATS shares a sequence identity of 28 % with FtmPT1, which catalyses the prenylation of the cyclic dipeptide cyclo-L-Trp-L-Pro at the C-2 position of the indole moiety (Grundmann & Li, 2005), and 26 % with FgaPT1, which prenylates fumigaclavine A at the C-2 position of the indole moiety (Unsöld & Li, 2006). 7-DMATS accepted Trp, but not Tyr as a substrate. However, with the exception of its orthologues in N. fischeri NRRL181 and A. terreus NIH2624, with sequence identities of 95 and 82 % (Table 2, see below) at the amino acid level, respectively, 7-DMATS showed the highest sequence similarity to SirD from the biosynthetic gene cluster of sirodesmin from L. maculans (Gardiner et al., 2004), i.e. a sequence identity of 34 % at the amino acid level. SirD has been proposed to catalyse the O-prenylation of L-Tyr, or its cyclic dipeptide, with Ser (Gardiner et al., 2005). It would be interesting to prove the function of this putative prenyltransferase biochemically.
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; Unsöld & Li, 2005, 2006), as well as DMATS in the biosynthesis of ergot alkaloids from Claviceps (Lee et al., 1976), Ca2+ enhanced the enzymic reaction of 7-DMATS slightly. This finding, together with the sequence similarity, provided experimental data for the hypothesis that the indole prenyltransferases from fungi belong to the same group of enzymes, and that they have evolved directly from a common ancestor.
Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of probably eight genes (Fig. 4, Table 2): two putative transcription factor genes Afu3g12940 and Afu3g12890, one putative transporter gene (Afu3g12900), one gene with unknown function (Afu3g12950), and four biosynthetic genes, i.e. the NRPS gene Afu3g12920, the prenyltransferase gene Afu3g12930 described in this study, a gene coding for a putative cytochrome P450 enzyme (Afu3g12960), and a putative methyltransferase gene Afu3g12910. With the exception of Afu3g12950, orthologous genes were also found in the genome sequence of N. fischeri NRRL181 and A. terreus NIH2624, with the same orientation and same relative position of the genes (GenBank entries) (Fig. 4). The gene products of the cluster from A. fumigatus share sequence identities of 92–96 % at the amino acid level with their orthologues from N. fischeri, and 73–88 % with the orthologues from A. terreus (Table 2), respectively. The gene Afu3g12950 in the gene cluster from A. fumigatus encodes a putative ubiquitin-conjugating enzyme. An orthologue of this gene was found in the gene cluster of N. fischeri, but not in the gene cluster of A. terreus. Therefore, it is questioned whether Afu3g12950 is essential for the biosynthesis of the compound encoded by this gene cluster.
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; Nierman et al., 2005). Deletion of the NRPS gene Afu3g12920 from the genome of A. fumigatus CEA17
akuB (Silva Ferreira et al., 2006) by T. Heinekamp and A. A. Brakhage (HKI, Jena, Germany), and HPLC analysis of the cultural extracts by our group, showed that no changes of the profile of the secondary metabolite production could be observed in the obtained mutants, in comparison with that of the wild-type (data not shown). This indicated that one or more genes of this cluster were not expressed, or were expressed at very low levels, so that no product of this cluster accumulated in the wild-type, and, therefore, no effect on secondary metabolite production could be observed after deletion of a gene from the cluster. This finding is in accordance with the results reported by Cramer et al. (2006b) that Afu3g12920 is not expressed, or is expressed at very low levels, under the conditions tested in their study. However, it can not be excluded that this cluster could be expressed in situ after inhalation of the fungus, and the secondary metabolite may then play a role as virulence factor.
Comparison with database entries revealed that the four biosynthesis enzymes EAL92287, EAL92290 (7-DMATS), EAL92291 and EAL92292 share sequence identities of 39, 34, 36 and 38 % with SirC, SirD, SirP and SirM, respectively (Table 2), which have been identified in the gene cluster of sirodesmin, which is an ETP from L. maculans (Gardiner et al., 2004). Sequence identities of 32, 29 and 34 % were also observed between EAL92287 and GliC, EAL92291 and GliP, as well as EAL92293 and GliM, respectively (Table 2). GliP, GliC and GliM are members of the gene cluster of the ETP gliotoxin from A. fumigatus (Gardiner & Howlett, 2005). The sequence identities of EAL92294 to SirZ and GliZ were found to be 23 and 27 %, respectively (Table 2). Therefore, the cluster discussed here is proposed to function in the biosynthesis of an ETP derivative (Cramer et al., 2006b; Nierman et al., 2005).
However, database and literature searches revealed that none of the known ETP derivatives contain a prenylated Trp moiety (Gardiner et al., 2005). Also, and more importantly, a homologue of the putative thioredoxin reductase, GliT/SirT, in the gene cluster of gliotoxin/sirodesmin, which is proposed to be responsible for the formation of the disulphide bond of the ETP derivatives (Gardiner et al., 2005; Gardiner & Howlett, 2005), is absent in the cluster. Therefore, it could be speculated that the cluster discussed here is not intact, or that the product of the gene cluster has a structure other than an ETP or a derivative thereof. Considering the fact that the cluster is present in the three fungi mentioned above, it seems that the latter is the most plausible explanation. The product of the gene cluster could be a derivative of a cyclic dipeptide containing 7-DMAT. It is plausible that L-Trp is converted to 7-DMAT, and that this is catalysed by the prenyltransferase 7-DMATS characterized in this study. 7-DMAT would then undergo the condensation with a second amino acid, which could not be predicted by the sequence analysis, to a prenylated (cyclic) dipeptide, under the catalysis of the putative NRPS EAL92291. Like GliP, EAL92291 has a trimodular architecture (A1-T1-C1-A2-T2-C2-T3) (Balibar & Walsh, 2006). This is in contrast to that of SirP of the sirodesmin cluster, and FtmPS for the formation of cyclo-L-Trp-L-Pro from the fumitremorgin cluster, as these do not contain the third thiolation domain T3 (Gardiner et al., 2004; Maiya et al., 2006). It has been proven by biochemical investigation that the A1 and A2 domains in GliP are responsible for the activation of L-Phe and L-Ser, respectively, which are then loaded onto the T1 and T2 domains of GliP, respectively. The linear enzyme-bond peptide L-Phe-L-Ser-T2 was formed under the catalysis of the condensation domain C1 (Balibar & Walsh, 2006). The roles of the second condensation domain C2, and the third thiolation domain T3, are unclear, although the results of mutational experiments have suggested that they are involved in the same process (Balibar & Walsh, 2006). It has been speculated that the subsequent reactions to gliotoxin, e.g. methylation or sulphur insertion, may occur while the linear dipeptide is still covalently bound to the NRPS GliP (Balibar & Walsh, 2006). The release of an intermediate in the biosynthesis could occur at some later step. This could also be the case for EAL92291. This means that an unmodified cyclic dipeptide with the prenylated tryptophan is probably not involved in the biosynthesis of the unknown compound discussed here. The putative methyltransferase EAL92290 is probably responsible for the N-methylation of an enzyme-bound linear dipeptide or the diketopiperazine ring, similar to the proposed function of GliM and SirM in the biosynthesis of gliotoxin and sirodesmin (Gardiner & Howlett, 2005). The putative cytochrome P450 EAL92287 could be involved in the modification of the enzyme-bond dipeptide or a derivative thereof. The two regulators EAL92289 and EAL92294 are probably involved in the regulation, and the putative transporter EAL92293 could be responsible for the efflux, of the product of the gene cluster from the fungus. Deletion of the genes from the genome of N. fischeri or A. terreus, in which this gene cluster is expressed, would explore the natural role of the gene cluster.
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ACKNOWLEDGEMENTS |
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Edited by: B. A. Horwitz
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REFERENCES |
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Received 12 April 2007;
revised 14 May 2007;
accepted 17 May 2007.
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