Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
Correspondence
Shu-Ming Li
shuming.li{at}uni-tuebingen.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Recently, the whole genome of A. fumigatus (strain AF293) was sequenced by an international consortium with The Wellcome Trust Sanger Institute (UK, http://www.sanger.ac.uk/Projects/A_fumigatus/), The Institute for Genomic Research (USA; http://www.tigr.org/tdb/e2k1/afu1/) and other institutions. From the available, but not yet annotated, genome sequence of A. fumigatus, we could identify a DMATS gene, fgaPT2. Here we report the cloning and heterologous expression of this gene, followed by the purification and biochemical characterization of the DMATS (systematic name dimethylallyl diphosphate : L-tryptophan dimethylallyltransferase; EC 2.5.1.34) that it encodes.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial and fungal strains, plasmids and culture conditions.
The Escherichia coliSacc. cerevisiae shuttle vector pYES2/NT B and Sacc. cerevisiae INVSc1 were obtained from Invitrogen. pGEMT and pSL1180 were obtained from Promega and Amersham Biosciences, respectively. Sacc. cerevisiae INVSc1 was maintained on Yeast Extract Peptone Glucose Medium (Ausubel et al., 1996). E. coli XL-1 Blue MRF' (Stratagene) and DH5
(Invitrogen) were used for cloning experiments, and were grown in liquid Luria-Bertani (LB) or on solid LB medium with 1·5 % agar at 37 °C (Sambrook & Russell, 2001
). Carbenicillin (50 µg ml1) was used for selection of recombinant E. coli strains.
BAC clone AfA 32C2 containing the genomic DNA of A. fumigatus strain AF293 was kindly provided by David Harris of the Wellcome Trust Sanger Institute (Cambridge, UK), and used as genomic DNA template for PCR amplification. 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.
DNA isolation, manipulation and cloning.
Standard procedures for DNA isolation and manipulation were performed as described by Sambrook & Russell (2001). Isolation of BAC DNA and plasmids from E. coli was carried out with ion-exchange columns (Nucleobond AX kits; Macherey-Nagel), according to the manufacturer's protocol.
Overproduction and purification of FgaPT2 protein.
For construction of the expression plasmid pIU11, two overlapping fragments of the coding sequences of fgaPT2 were amplified independently of each other. A genuine NcoI restriction enzyme recognition site is located in the overlapping part of both fragments. The fragment of 1164 bp at the 5' end was amplified from genomic DNA on BAC AfA32C2 by using the primers PT2-561_for (5'-CCAAGATGGATCCCATGAAGGCAGCCAATG-3') and PT2-1720_rev (5'-AGTACGACTTCAAAGTAGTTTCGTAGGTGC-3'). Bold letters represent mutations inserted in comparison to the original sequence to give the underlined BamHI restriction site. The fragment of 624 bp at the 3' end was amplified from cDNA by using the primers PT2-1375_for (5'-CTAGAAGCCATGGAGGACCTGTGGACTCTG-3'), and PT2-2133_rev (5'-CGCGGAATTCATCGGGTTACAGCCCGGAA-3'). Bold letters represent mutations inserted in comparison to the original sequence to give the underlined EcoRI restriction site. Both fragments were ligated into pGEMT to give pIU3 containing the fragment at the 5' end, and pIU4 containing the fragment at the 3' end. pIU3 was digested with BamHI and NcoI, and the obtained BamHINcoI fragment of 818 bp was ligated into pSL1180, which had been restricted with BamHI and NcoI, to give pIU5. pIU4 was digested with NcoI and EcoRI, and the obtained NcoIEcoRI fragment of 607 bp was ligated into pIU5, which had also been digested with NcoI and EcoRI, to give pIU7. The resulting plasmid containing the complete coding sequence of fgaPT2 was sequenced to identify potential errors by PCR amplification (MWG-Biotech). The whole coding sequence of fgaPT2 was released from pIU7 by digestion with BamHI and EcoRI, and ligated into the expression vector pYES2/NT B, which had been restricted with the same enzymes, to give the expression construct pIU11.
pIU11 was introduced into Sacc. cerevisiae INVSc1 by electroporation (Ausubel et al., 1996). Recombinants were selected and maintained on minimal medium lacking uracil. For gene expression, the cells were grown in baffled 3000 ml Erlenmeyer flasks containing 300 ml liquid minimal medium (with 2 % glucose) at 30 °C and 200 r.p.m. for 24 h. Then the cells were transferred into medium containing 1 % raffinose and 2 % galactose for induction, and cultivated for a further 16 h before harvest. Total protein was obtained after breaking of the cells using glass beads (Ausubel et al., 1996
) in 50 mM sodium phosphate buffer (pH 7·4) with 5 % (v/v) glycerol and 1 mM PMSF. 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 instruction.
Protein analysis.
Standard protein techniques were used as described by Bradford (1976) and Laemmli (1970)
. The molecular mass of native FgaPT2 was determined by gel filtration on a HiLoad 26/60 Superdex 200 column (Amersham Biosciences) using 50 mM Tris/HCl buffer (pH 8·0) containing 150 mM NaCl. The column was calibrated with dextran blue 2000 (2000 kDa), aldolase (158 kDa), albumin (66 kDa), ovalbumin (45 kDa) and ribonuclease A (13·7 kDa) (Amersham Biosciences).
Assay for DMATS activity.
For quantitative determination of enzyme activity, the reaction mixture (100 µl) contained 50 mM Tris/HCl (pH 7·5), 5 mM CaCl2, 1 mM L-tryptophan, 1 mM DMAPP and 0·11 µg purified FgaPT2. After incubation for 10 min at 30 °C, the reaction was stopped with 10 µl TCA (1·5 M). The protein was removed by centrifugation at 13 000 g for 10 min. Enzymic products were analysed by HPLC at 269 nm using a Multosphere RP 18-5 column (250x4 mm, 5 µm; C+S Chromatographie Service) at a flow rate of 1 ml min1. A linear gradient of 2070 % acetonitrile in 0·1 % aqueous trifluoroacetic acid in 20 min was used. Authentic DMAT (Yokoyama) (Hikawa et al., 2000) was used as a standard. All quantitative enzyme activity data in this manuscript are mean values from two independent enzyme assays.
Preparative enzymic synthesis and structural elucidation of dimethylallyltryptophan and derivatives.
To a 1·5 ml glass vial, DMAPP (1 mM final concentration), L-tryptophan, 5-methyl-DL-tryptophan or 6-methyl-DL-tryptophan (1 mM), CaCl2 (5 mM), Tris/HCl (50 mM, pH 7·5) and FgaPT2 (75 µg) were added to a final volume of 1 ml. The mixtures were incubated at 30 °C for 16 h. A 100 µl volume of TCA (1·5 M) was added, and the protein was removed by centrifugation. 4-Dimethylallyltryptophan (DMAT) and derivatives were purified by HPLC using the method described above. The products obtained (0·050·2 mg) were analysed by 1H-NMR spectroscopy, H-H-COSY-NMR and positive-ion fast atom bombardment (FAB)-MS.
Nucleotide sequence accession number.
The nucleotide sequences of the genomic DNA reported in this study are available at The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/A_fumigatus/). The cDNA sequence of fgaPT2 is available at GenBank under accession number AY775787.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The genomic sequence of this putative prenyltransferase gene consists of three exons of 1151, 122 and 104 bp, interrupted by two introns of 61 and 74 bp. The existence of these introns was confirmed by sequencing of a PCR fragment amplified with cDNA as the template (see below). TATA and CCAAT boxes were found 68 and 132 bp upstream of the putative start codon of fgaPT2, respectively. The start codon and the introns were predicted by alignment with DMATS sequences from other fungi (Tsai et al., 1995; Tudzynski et al., 1999
; Wang et al., 2004
) in combination with the prediction obtained from FGENESH.
The predicted gene product of fgaPT2 comprises 459 amino acids, and has a calculated molecular mass of 52·5 kDa. FgaPT2 shows high similarity to putative DMATS from various fungi. For example, FgaPT2 has an identity of 60 % to DMATS from Neotyphodium coenophialum (Wang et al., 2004), 54 % to DMATS from C. purpurea (Tudzynski et al., 1999
), 56 % to DMATS from Balansia obtecta (Wang et al., 2004
), and 52 % to DMATS from C. fusiformis (Tsai et al., 1995
). The prenyl diphosphate binding site (ND)DXXD (Liang et al., 2002
) was not found in the sequence of FgaPT2.
Cloning of fgaPT2
To eliminate the two introns in the sequence of fgaPT2, a fragment of 624 bp at the 3' end was amplified using PCR from cDNA of A. fumigatus B5233, which is available in the form of phagemids isolated from a cDNA library, whereas a 5'-end fragment of 1164 bp was amplified from genomic DNA of A. fumigatus AF293 in the form of BAC DNA. The two PCR fragments overlap by 346 bp, and were cloned via a genuine NcoI recognition site in the overlapping region into a cloning vector. Sequencing of the resulting plasmid revealed one nucleotide difference from the published genome sequence of strain AF293, i.e. G instead of T at position 1327 of the coding sequence, which leads to an exchange of serine by alanine in position 443 in FgaPT2. PCR fragments in four clones from two independent amplifications show the same sequence at this position, suggesting that the difference may not be due to an error of PCR amplification. Rather, the sequence of the cDNA library obtained from A. fumigatus strain B5233 may differ from the published genomic sequence of A. fumigatus AF293.
Overproduction and purification of FgaPT2
The coding sequence of fgaPT2 was cloned into the vector pYES/NT B. Soluble proteins obtained from transformants of S. cerevisiae harbouring the expression construct were used for purification with Ni-NTA agarose, and FgaPT2 was purified to apparent homogeneity, as judged by SDS-PAGE (Fig. 2a). The observed molecular mass was 57 kDa, and corresponds very well to the calculated mass of 56 kDa for His6-FgaPT2. For the His-tagged protein, a protein yield of 0·2 mg pure FgaPT2 per litre of culture was obtained, which was low, but sufficient for the biochemical characterization.
|
The enzymic product was subsequently isolated on a preparative scale (see Methods) and subjected to NMR and MS analysis.
Comparison of the 1H-NMR spectrum of the isolated product with that of L-tryptophan (Table 1) revealed that in the spectrum of the isolated compound the doublet at 7·71 p.p.m. for H-4 of L-tryptophan had disappeared. Instead, additional signals for a dimethylallyl moiety were observed at 5·37 (br t, 7·2 Hz, H-2'), 3·79 (d, 7·2 Hz, H-1'), 1·79 (s, 3H-5') and 1·76 (s, 3H-4') p.p.m., respectively. The expected correlations of these protons were proven by a H-H-COSY spectrum. Positive FAB-MS showed an ion at m/z 273 ([M+H]+) confirming the presence of a prenylated tryptophan. The NMR and MS data (m/z (intensity): 93 (58·0), 198 (41·6), 217 (20), 256 (72), 273 (100) [M+H]+, 295 (8) [M+Na]+) proved that the enzymic product is the expected DMAT (Fig. 2b
). These data correspond to those of DMAT reported by Gebler & Poulter (1992)
, except for a 0·25 p.p.m. difference for H-
of DMAT. The chemical shift of this proton is dependent on the pH value of the NMR samples. The spectrum of the isolated compound was taken at pH 2, whereas the literature data were obtained at pH 4·3. For direct comparison, we recorded the 1H-NMR spectrum of L-tryptophan at two different pH values, i.e. pH 2 vs pH 4·3. The signal of H-
of tryptophan was observed at 4·14 p.p.m. at pH 2, whereas an upfield shift to 4·04 p.p.m. was found when the sample was measured at pH 4·3. This corresponds to the downfield shift observed for H-
of DMAT at pH 2 compared to the literature data (Gebler & Poulter, 1992
).
|
The FgaPT2 reaction apparently followed MichaelisMenten kinetics, and the Km values were determined as 8 µM for L-tryptophan, and 4 µM for DMAPP. These data are similar to those reported by Cress et al. (1981) for purified DMATS from Claviceps sp. SD58, i.e. 8·8 and 7·2 µM for L-tryptophan and DMAPP, respectively, but are at variance with those reported by Lee et al. (1976)
, namely Km values of 67 and 200 µM for L-tryptophan and DMAPP, respectively. The maximum reaction velocity observed with FgaPT2 was 0·198 µmol min1 mg1, corresponding to a turnover number of 0·37 s1.
FgaPT2 was found to be specific for its substrates L-tryptophan and DMAPP. Low product formation was observed when D-tryptophan (1·8 % of that with L-tryptophan), 5-methyl-DL-tryptophan (11·6 %) or 6-methyl-DL-tryptophan (6·8 %) was used instead of L-tryptophan. The enzymic product of 6-methyltryptophan was identified unequivocally as 4-dimethylallyl-6-methyltryptophan (6-methyl-DMAT) by 1H-NMR (Table 1) and MS analysis (m/z (intensity): 212 (100), 231 (34), 270 (66), 287 (76) [M+H]+). 5-Methyltryptophan was converted by FgaPT2 to dimethylallyl-5-methyltryptophan, as confirmed by MS analysis (m/z (intensity): 212 (85), 231 (31), 270 (65), 287 (100) [M+H]+, 309 (32) [M+Na]+). There was no product formation (<0·2 %) observed with 4-hydroxybenzoic acid, the substrate of the prenyltransferase of ubiquinone biosynthesis (Melzer & Heide 1994
), or with 4-hydroxyphenylpyruvic acid or umbelliferone, the substrates of previously identified prenyltransferases of secondary metabolism (Hamerski et al., 1990
; Pojer et al., 2003
). These results are comparable with those obtained with DMATS from C. purpurea (Lee et al., 1976
). When DMAPP was replaced with geranyl diphosphate, very low product formation (0·7 % of that with DMAPP) was observed. A low product formation was also observed with isopentenyl diphosphate (3·4 % of that with DMAPP).
Using gel chromatography, the native molecular mass of FgaPT2 was determined as 8691 kDa, suggesting that the protein is active as a dimer.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FgaPT2 is the first enzyme involved in ergot alkaloid biosynthesis to be heterologously overproduced and purified to homogeneity. Just like DMATS from C. purpurea (Gebler & Poulter, 1992), CloQ from Strep. roseochromogenes (Pojer et al., 2003
) and LtxC from L. majuscula (Edwards & Gerwick, 2004
), FgaPT2 from A. fumigatus is active in the complete absence of divalent cations, and does not contain the prenyl diphosphate binding motif (N/D)DXXD of trans-prenyltransferases such as farnesyl diphosphate (FPP) synthase (Liang et al., 2002
). The substrate binding sites of CloQ, LtxC and DMATS are unknown. All three enzymes catalyse the C-prenylation of an aromatic nucleus, and since they do not require Mg2+ or other divalent metal ions for activity, they apparently differ from FPP synthase in substrate binding and catalytic mechanism (Liang et al., 2002
). FgaPT2 shows significant sequence homology only to DMATS from fungi (Tsai et al., 1995
; Tudzynski et al., 1999
; Wang et al., 2004
), but not to prenyltransferases from bacteria, e.g. CloQ and LtxC. Therefore the aromatic prenyltransferases from fungi and bacteria may represent different evolutionary groups of prenyltransferases. Information on additional fungal prenyltransferases could support this hypothesis.
Further analysis of the genome sequence of A. fumigatus in the vicinity of fgaPT2 revealed the presence of an additional homologue of cpd1, termed fgaPT1, which showed an identity of 32 % to cpd1 at the amino acid level. fgaPT1 and fgaPT2 are separated by only 10 kb (Fig. 3a). Interestingly, we could identify four additional ORFs, termed fgaOX1, fgaOX2, fgaOX3 and fgaCAT, in the vicinity of fgaPT1 and fgaPT2 (Fig. 3
), which showed high sequence similarity to genes from the biosynthetic gene cluster of ergot alkaloids from C. purpurea (Fig. 3
), i.e. to cpox1, cpox2, cpox3 and cpcat2 (Correia et al., 2003
). Only part of the ergot alkaloid cluster of C. purpurea is published, and therefore additional similarities between this cluster and the genomic region surrounding fgaPT2 in A. fumigatus may still be identified. Together with the DMATS, the four genes mentioned above may be involved in the formation of the clavine skeleton, the common structural feature of ergot alkaloids from both organisms (Fig. 1
). Fumigaclavine C has an additional prenyl moiety at C-2 of the indole nucleus, which could account for the presence of the second putative prenyltransferase gene fgaPT1. No homologue for the non-ribosomal peptide synthetase gene cpps1 (Fig. 3
) of the ergot alkaloid cluster in C. purpurea (Tudzynski et al., 1999
) was found in the vicinity of fgaPT2, consistent with the absence of a peptide moiety in fumigaclavines (Fig. 1
). Therefore, the genes surrounding fgaPT2 in the A. fumigatus genome may possibly represent a biosynthetic gene cluster for fumigaclavines and their structural analogues elymoclavine and festuclavine (Fig. 1
) (Flieger et al., 1997
; Spilsbury & Wilkinson, 1961
; Yamano et al., 1962
). Future functional investigations of the genes in this putative biosynthetic gene cluster may enhance the understanding of the biosynthesis of the pharmaceutically important ergot alkaloids.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Correia, T., Grammel, N., Ortel, I., Keller, U. & Tudzynski, P. (2003). Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea. Chem Biol 10, 12811292.[CrossRef][Medline]
Cress, W. A., Chayet, L. T. & Rilling, H. C. (1981). Crystallization and partial characterization of dimethylallyl pyrophosphate : L-tryptophan dimethylallyltransferase from Claviceps sp. SD58. J Biol Chem 256, 1091710923.
Edwards, D. J. & Gerwick, W. H. (2004). Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase. J Am Chem Soc 126, 1143211433.[CrossRef][Medline]
Flieger, M., Wurst, M. & Shelby, R. (1997). Ergot alkaloids sources, structures and analytical methods. Folia Microbiol 42, 329.
Floss, H. G. (1976). Biosynthesis of ergot alkaloids and related compounds. Tetrahedron 32, 873912.[CrossRef]
Gebler, J. C. & Poulter, C. D. (1992). Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea. Arch Biochem Biophys 296, 308313.[CrossRef][Medline]
Gröger, D. & Floss, H. G. (1998). Biochemistry of ergot alkaloids achievements and challenges. In Alkaloids: Chemistry and Biology, vol. 50, pp. 171218. Edited by G. A. Cordell. San Diego: Academic Press.
Hamerski, D., Schmitt, D. & Matern, U. (1990). Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures. Phytochemistry 29, 11311135.[CrossRef][Medline]
Hikawa, H., Yokoyama, Y. & Murakami, Y. (2000). A short synthesis of optically active ,
-dimethylallyltryptophan (DMAT). Synthesis 214216.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
Lee, S. L., Floss, H. G. & Heinstein, P. (1976). Purification and properties of dimethylallylpyrophosphate:tryptophan dimethylallyl transferase, the first enzyme of ergot alkaloid biosynthesis in Claviceps. sp. SD 58. Arch Biochem Biophys 177, 8494.[CrossRef][Medline]
Liang, P. H., Ko, T. P. & Wang, A. H. (2002). Structure, mechanism and function of prenyltransferases. Eur J Biochem 269, 33393354.
Melzer, M. & Heide, L. (1994). Characterization of polyprenyldiphosphate : 4-hydroxybenzoate polyprenyltransferase from Escherichia coli. Biochim Biophys Acta 1212, 93102.[Medline]
Pojer, F., Wemakor, E., Kammerer, B., Chen, H., Walsh, C. T., Li, S.-M. & Heide, L. (2003). CloQ, a prenyltransferase involved in clorobiocin biosynthesis. Proc Natl Acad Sci U S A 100, 23162321.
Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Spilsbury, J. F. & Wilkinson, S. (1961). Isolation of festuclavine and two new clavine alkaloids from Aspergillus fumigatus. J Chem Soc 20852091.
Tsai, H. F., Wang, H., Gebler, J. C., Poulter, C. D. & Schardl, C. L. (1995). The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis. Biochem Biophys Res Commun 216, 119125.[CrossRef][Medline]
Tudzynski, P., Holter, K., Correia, T., Arntz, C., Grammel, N. & Keller, U. (1999). Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol Gen Genet 261, 133141.[CrossRef][Medline]
Tudzynski, P., Correia, T. & Keller, U. (2001). Biotechnology and genetics of ergot alkaloids. Appl Microbiol Biotechnol 57, 593605.[CrossRef][Medline]
Wang, J., Machado, C., Panaccione, D. G., Tsai, H. F. & Schardl, C. L. (2004). The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass. Fungal Genet Biol 41, 189198.[CrossRef][Medline]
Williams, R. M., Stocking, E. M. & Sanz-Cervera, J. F. (2000). Biosynthesis of prenylated alkaloids derived from tryptophan. Topics Curr Chem 209, 97173.
Yamano, T., Kishino, K., Yamatodani, S. & Abe, M. (1962). Ergot fungus. XLIX. Investigation on ergot alkaloids found in cultures of Aspergillus fumigatus. Takeda Kenkyusho Nenpo 21, 95101.
Received 9 November 2004;
revised 18 January 2005;
accepted 8 February 2005.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |