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Combinatorial Assembly of Simple and Complex d-Lysergic Acid Alkaloid Peptide Classes in the Ergot Fungus Claviceps purpurea

2009; Elsevier BV; Volume: 284; Issue: 11 Linguagem: Inglês

10.1074/jbc.m807168200

1083-351X

Ingo Ortel, Ullrich Keller,

Mycorrhizal Fungi and Plant Interactions

The ergot fungus Claviceps purpurea produces both ergopeptines and simple d-lysergic acid alkylamides. In the ergopeptines, such as ergotamine, d-lysergic acid is linked to a bicyclic tripeptide in amide-like fashion, whereas in the d-lysergylalkanolamides it is linked to an amino alcohol derived from alanine. We show here that these compound classes are synthesized by a set of three non-ribosomal lysergyl peptide synthetases (LPSs), which interact in a combinatorial fashion for synthesis of the relevant product. The trimodular LPS1 assembles with LPS2, the d-lysergic acid recruiting module, to synthesize the d-lysergyltripeptide precursors of ergopeptines from d-lysergic acid and the three amino acids of the peptide chain. Alternatively, LPS2 can assemble with a distinct monomodular non-ribosomal peptide synthetase (NRPS) subunit (ergometrine synthetase) to synthesize the d-lysergic acid alkanolamide ergometrine from d-lysergic acid and alanine. The synthesis proceeds via covalently bound d-lysergyl alanine and release of dipeptide as alcohol with consumption of NADPH. Enzymatic and immunochemical analyses showed that ergometrine synthetase is most probably the enzyme LPS3 whose gene had been identified previously as part of the ergot alkaloid biosynthesis gene cluster in C. purpurea. Inspections of all LPS sequences showed no recognizable peptide linkers for their protein-protein interactions as in NRPS subunits of bacteria. Instead, they all carry conserved N-terminal domains (C0-domains) with similarity to the C-terminal halves of NRPS condensation domains pointing to an alternative mechanism of subunit-subunit interactions in fungal NRPS systems. Phylogenetic analysis of LPS modules and the C0-domains suggests that these enzyme systems most probably evolved by module duplications and rearrangements from a bimodular ancestor. The ergot fungus Claviceps purpurea produces both ergopeptines and simple d-lysergic acid alkylamides. In the ergopeptines, such as ergotamine, d-lysergic acid is linked to a bicyclic tripeptide in amide-like fashion, whereas in the d-lysergylalkanolamides it is linked to an amino alcohol derived from alanine. We show here that these compound classes are synthesized by a set of three non-ribosomal lysergyl peptide synthetases (LPSs), which interact in a combinatorial fashion for synthesis of the relevant product. The trimodular LPS1 assembles with LPS2, the d-lysergic acid recruiting module, to synthesize the d-lysergyltripeptide precursors of ergopeptines from d-lysergic acid and the three amino acids of the peptide chain. Alternatively, LPS2 can assemble with a distinct monomodular non-ribosomal peptide synthetase (NRPS) subunit (ergometrine synthetase) to synthesize the d-lysergic acid alkanolamide ergometrine from d-lysergic acid and alanine. The synthesis proceeds via covalently bound d-lysergyl alanine and release of dipeptide as alcohol with consumption of NADPH. Enzymatic and immunochemical analyses showed that ergometrine synthetase is most probably the enzyme LPS3 whose gene had been identified previously as part of the ergot alkaloid biosynthesis gene cluster in C. purpurea. Inspections of all LPS sequences showed no recognizable peptide linkers for their protein-protein interactions as in NRPS subunits of bacteria. Instead, they all carry conserved N-terminal domains (C0-domains) with similarity to the C-terminal halves of NRPS condensation domains pointing to an alternative mechanism of subunit-subunit interactions in fungal NRPS systems. Phylogenetic analysis of LPS modules and the C0-domains suggests that these enzyme systems most probably evolved by module duplications and rearrangements from a bimodular ancestor. d-Lysergic acid (see Fig. 1, I) is the pharmacophore of the various amide/peptide-type ergot alkaloids. In these compounds, the carboxyl group of d-lysergic acid is amidated with simple amino alcohols or small peptide chains, which, depending on their structures, confer the tetracylic methylergolene skeleton of d-lysergic acid similarity to different neurotransmitters such as dopamine, serotonine, or adrenaline (1Stadler P.A. Giger R. Krosgard-Larson P. Kofod H. Natural Products and Drug Development. Munksgaard, Copenhagen, Denmark1984: 463-485Google Scholar). Several d-lysergic acid alkaloids or synthetic derivatives are used in the treatment of a number of disorders in the vascular and central nervous systems (2Burkhalter A. Julius D.J. Katzung B.G. Katzung B.G. Basic and Clinical Pharmacology. Appleton-Lange, New York1998: 261-286Google Scholar, 3Berde B. Stuermer E. Aellig W.H. Berde B. Schild O.H. Introduction to the Pharmacology of Ergot Alkaloids and Related Compounds. Springer, Berlin1978: 1-28Google Scholar). The natural d-lysergic acid amides are produced by a wide variety of ascomycete fungi, mostly belonging to the family Clavicipitaceae (4Flieger M. Wurst M. Shelby R. Folia Microbiol. 1997; 42: 3-29Crossref PubMed Scopus (137) Google Scholar). Most prominent among these is Claviceps purpurea, which grows on cereals and forms there the sclerotia known as ergot, which has long been the main source of these compounds (5Kobel H. Sanglier J.J. Rehm H.J. Reeds G. Biotechnology. VCH Verlagsgesellschaft, Weinheim, Germany1986: 569-609Google Scholar). Remarkably, d-lysergic acid rarely occurs in free form in these fungi and, like its biosynthetic precursors, the clavine alkaloids agroclavine and elymoclavine, has little biological activity (6Klotz J.L. Bush L.P. Smith D.L. Schafer W.D. Smith L.L. Vevoda A.O. Craig A.M. Arrington B.C. Strickland J.R. J. Animal Sci. 2006; 84: 3167-3175Crossref PubMed Scopus (44) Google Scholar). In the ergopeptines (see Fig. 1, II) d-lysergic acid is amidated with bicyclic tripeptide chains. The first two amino acid positions of the tripeptide chains are normally occupied by non-polar amino acids. The third amino acid is nearly always proline. Biosynthetically, the ergopeptines are derived from the l-ergopeptams (d-lysergyl peptide lactams, see Fig. 1, III), structurally identical to ergopeptines except that the cyclol bridge between the α-C of the first amino acid and the carboxyl group of proline is missing (7Stadler P.A. Planta Med. 1982; 46: 131-144Crossref PubMed Scopus (22) Google Scholar). l-Ergopeptams have, like the ergopeptines, l-proline, whereas the d-ergopeptams, which in ergot fungi sometimes occur as co-products with ergopeptines, have d-proline. d-Ergopeptams are considered as dead-end products of the alkaloid peptide pathway arising from spontaneous isomerization of l-ergopeptams prior to their conversion to ergopeptines, the regular end products of the alkaloid pathway (7Stadler P.A. Planta Med. 1982; 46: 131-144Crossref PubMed Scopus (22) Google Scholar). The l-ergopeptams are assembled non-ribosomally from d-lysergic acid and the amino acids of the peptide chain by the lysergyl peptide synthetases (LPS1 and LPS2) 2The abbreviations used are: LPS, d-lysergylpeptide synthetase; NRPS, nonribosomal peptide synthetase; C0-domains, modified N-terminal C-domains; lpsCred, gene fragment encoding a portion of LPS3 Red-domain; CTS, C. purpurea putative toxin synthetase; M, module; AT, adenylationthiolation didomain. (8Keller U. Han M. Stoeffler-Meilicke M. Biochemistry. 1988; 27: 6164-6170Crossref PubMed Scopus (23) Google Scholar, 9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 10Walzel B. Riederer B. Keller U. Chem. Biol. 1997; 4: 223-230Abstract Full Text PDF PubMed Scopus (52) Google Scholar). The monomodular LPS2 recruits d-lysergic acid, the formal N terminus of the chain, as a thioester. d-Lysergic acid is then progressively elongated to the d-lysergyl-mono-, -di-, and -tripeptides by the trimodular LPS1 (see Fig. 2a). Enzyme-bound d-lysergyl tripeptide is finally released as l-ergopeptam. LPS1 and LPS2 have sizes of 370 and 141 kDa, respectively, and are a rare example of fungal NRPS systems composed of subunits (9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Their genes have been sequenced and analyzed (11Tudzynski P. Hölter K. Correia T. Arntz C. Grammel N. Keller U. Mol. Gen. Genet. 1999; 261: 133-141Crossref PubMed Scopus (147) Google Scholar, 12Correia T. Grammel N. Ortel I. Keller U. Tudzynski P. Chem. Biol. 2003; 10: 1281-1292Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Inspection of other ergot fungi-producing ergopeptines by DNA sequencing has revealed both LPS1 and LPS2 gene orthologues (designated lpsA and lpsB) in the respective genomes and also that in C. purpurea the lpsA gene may occur in duplicate (13Panaccione D.G. Johnson R.D. Wang J. Young C.A. Damrongkool P. Scott B. Schardl C.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12820-12825Crossref PubMed Scopus (143) Google Scholar, 14Damrongkool P. Sedlock A.B. Young C.A. Johnson R.D. Goetz K.E. Scott B. Schardl C.L. Panaccione D.G. DNA Seq. 2005; 16: 379-385Crossref PubMed Scopus (11) Google Scholar, 15Fleetwood D.J. Scott B. Lane G.A. Tanaka A. Johnson R.D. Appl. Environ. Microbiol. 2007; 73: 2571-2579Crossref PubMed Scopus (102) Google Scholar, 16Haarmann T. Machado C. Lübbe Y. Correia T. Schardl C.L. Panaccione D.G. Tudzynski P. Phytochemistry. 2005; 66: 1312-1320Crossref PubMed Scopus (95) Google Scholar). Both lpsAs and lpsBs have been found in the ergot fungi so far analyzed to be co-localized with most if not all genes encoding the steps of clavine alkaloid biosynthesis, including the conversion of elymoclavine to d-lysergic acid forming an ergot alkaloid biosynthesis cluster (11Tudzynski P. Hölter K. Correia T. Arntz C. Grammel N. Keller U. Mol. Gen. Genet. 1999; 261: 133-141Crossref PubMed Scopus (147) Google Scholar, 15Fleetwood D.J. Scott B. Lane G.A. Tanaka A. Johnson R.D. Appl. Environ. Microbiol. 2007; 73: 2571-2579Crossref PubMed Scopus (102) Google Scholar, 17Haarmann T. Ortel I. Tudzynski P. Keller U. Chembiochem. 2006; 7: 645-652Crossref PubMed Scopus (54) Google Scholar, 18Panaccione D.G. Tapper B.A. Lane G.A. Davies E. Fraser K.J. Agric. Food Chem. 2003; 51: 6429-6437Crossref PubMed Scopus (46) Google Scholar). In the simple d-lysergic acid amides such as ergometrine (synonymous with ergonovine and ergobasine) (Fig. 1, IV) and d-lysergic acid hydroxyethylamide, d-lysergic acid is amidated with alaninol or ethanolamine, respectively. Ergometrine has a pronounced uterotonic effect compared with ergotamine as revealed by the finding that pure ergotamine acted more slowly than when aqueous extracts of ergot were administered (19De Costa C. Lancet. 2002; 359: 1768-1770Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In field sclerotia of C. purpurea, the non-polar ergopeptines are always accompanied by these simple water-soluble d-lysergic acid alkylamides, indicating that the fungus augments the toxic effects by producing two different classes of d-lysergic acid alkaloids characterized by short and longer acting effects on the central nervous and vascular system (20Hofmann A. Die Mutterkornalkaloide. Enke Verlag, Stuttgart, Germany1964Google Scholar). In contrast to the ergopeptines, however, it was unknown how d-lysergic acid is assembled into that second class of ergot alkaloids represented by ergometrine nor which enzymes are involved. Strains and Cultures-Cultivation of strain Ecc93 of C. purpurea was described previously (8Keller U. Han M. Stoeffler-Meilicke M. Biochemistry. 1988; 27: 6164-6170Crossref PubMed Scopus (23) Google Scholar, 9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It produces mainly ergocristine with minor amounts of ergosine, ergotamine, and ergometrine (together with other low molecular weight alkaloids). Strain Ecc93 was routinely selected for maintenance of alkaloid high production as described previously (21Keller U. Appl. Environ. Microbiol. 1982; 46: 580-584Crossref Google Scholar). Chemicals and Radiochemicals-d-Lysergic acid was prepared by alkaline hydrolysis of ergotamine. Ergometrine was kindly donated by Dr. J. J. Sanglier, Novartis AG, Basel (Switzerland). Dihydrolysergic acid and dihydroergometrine were obtained by catalytic hydrogenation over palladium on charcoal of d-lysergic acid and ergometrine, respectively. Dihydrolysergylalanine was synthesized as described (10Walzel B. Riederer B. Keller U. Chem. Biol. 1997; 4: 223-230Abstract Full Text PDF PubMed Scopus (52) Google Scholar). [9,10-3H]Dihydroergocryptine (17.5 Ci/mmol) was from Hartmann Analytics, Braunschweig (Germany). l-[U-14C]Alanine (148 mCi/mmol), l-[U-14C]valine (256 mCi/mmol) and l-[U-14C]phenylalanine (460 mCi/mmol) were from Amersham Biosciences. [9,10-3H]Dihydrolysergic acid was prepared from [9,10-3H]dihydroergocryptine as described previously (8Keller U. Han M. Stoeffler-Meilicke M. Biochemistry. 1988; 27: 6164-6170Crossref PubMed Scopus (23) Google Scholar). Nucleic Acid Analysis-Extraction of chromosomal DNA of C. purpurea was with the PeqGold Fungal DNA Mini Kit according to the manufacturer's instructions (Peqlab, Erlangen, Germany). For PCR analysis, TaqDNA polymerase was used (Peqlab). Plasmid for subcloning of PCR fragments for sequencing was pCR4-TOPO (Invitrogen) and for expression of lpsCred was pQE30UA (Qiagen). Oligonucleotides for PCR amplification of portions of lpsC from C. purpurea Ecc93 were: IO_lps3red_for, 5′-GTGTTTGTGACAGGCGCCAGCGGATT; IO_lps3red_rev, 5′-CGGTTAAAGGATCCCCTATTACGAGGCC; IO_lps3C_for, 5′-CGAACACGCATCATTGTCTGCGC; IO_lps3C_rev, 5′-CAAGTGACCACACGAATACCCAGCC; IO_lps3A_for, 5′-GGATAACCCCTATGAATGATCGACCG; IO_lps3A_rev, 5′-CCTTTGAGAGAATGCTATGGAGGTTGG; IO_lps3Nterm_for, 5′-GTTTTATATGAGAACCAAGGTCCC; and IO_lps3Nterm_rev, 5′-GCAGACCTTCCCAGATAACACG. DNA sequencing was performed by AGOWA (Berlin, Germany). Sequences and multiple alignments were computed and arranged with ClustalW (22Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35075) Google Scholar) and Genedoc (23Nicholas K.B. Nicholas Jr., H.B. Deerfield D.W. EMBNEW NEWS. 1997; 4: 14Google Scholar). Cloning and Expression of lpsCred-The PCR fragment encoding a 192-amino acid-long region of the reductase domain of lpsC (accession number AJ884677) was obtained by amplifying a DNA fragment between nucleotides 3785 and 4361 of the lpsC gene using primers LPS3red_for/rev and genomic DNA from C. purpurea Ecc93 as template. The fragment was ligated by TOPO-cloning into vector pQE30UA (Qiagen), resulting in LpsCred_pQE30UA. Heterologous expression of plasmid LpsCred_pQE30UA in Escherichia coli M15 was in 1 liter of LB medium, 100 μg/ml ampicillin, and 25 μg/ml kanamycin at 37 °C. At an A600 of 0.6, induction was with 1 mm isopropyl-1-thio-β-d-galactopyranoside. Cells were harvested after further 4 h of incubation at 37 °C. Expression of LpsCred_pQE30UA resulted in the formation of inclusion bodies. For purification of LpsCred from E. coli M15 transformed with plasmid cpps3red_pQE30UA, 6 g of cells was resuspended in 0.05 m phosphate buffer (pH 8.0) containing 0.5 mg/ml lysozyme and passed through a French Press cell at 8,000 p.s.i. After incubation in the presence of 10 mm MgCl2 and 20 μg ml–1 DNase I (grade II, Sigma) for 30 min at room temperature, the suspension was centrifuged at 1500 × g for 30 min to separate the insoluble cellular fraction containing inclusion bodies. The collected fraction of inclusion bodies was resuspended in 0.05 m phosphate buffer (pH 8.0) containing 1% Triton X-100, disintegrated by ultrasonification (3 × 30 s, 50 watts) and centrifuged as before. The inclusion bodies were precipitated, and the procedure was repeated two times with buffers containing 0.5% Triton X-100 and finally buffer without Triton X-100. The inclusion bodies were dissolved in 0.05 m phosphate buffer (pH 8.0) containing 8 m urea and 4 mm dithiothreitol, and the resultant solution was centrifuged at 20,000 × g for 30 min. The clear supernatant containing denatured protein was dialyzed against 0.05 m phosphate buffer (pH 8.0) at 4 °C for 24 h, with two changes of buffer. The resulting suspension was centrifuged at 20,000 × g for 30 min. The supernatant containing the renatured protein was concentrated by ultrafiltration with Centricon YM10 membranes (Millipore, Schwalbach, Germany) and further purified by preparative SDS-PAGE (15%, 3-mm gel thickness) by the Laemmli method. Protein bands containing the recombinant protein were cut out from the gel and minced into small pieces for electroelution of protein overnight using a Biotrap BT 1000 (Schleicher & Schüll, Dassel, Germany) in 0.1 m Tris-HCl, 0.1% SDS, pH 8.0. Eluted protein was concentrated in a Centricon 30 microconcentrator (Amicon) to the desired concentration. Methods of Analysis-Techniques for amino acid analysis, radiomeasurements, and radiochemical analysis of enzymatically formed peptides have been described elsewhere (8Keller U. Han M. Stoeffler-Meilicke M. Biochemistry. 1988; 27: 6164-6170Crossref PubMed Scopus (23) Google Scholar, 9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). TLC solvent systems for amino acid analysis on Silica Gel 60 plates (Merck, Darmstadt, Germany) were isopropanol:acetic acid:water (7:3:2, by vol.) or butanol:acetic acid:water (4:2:2, by vol.). For separation of d-lysergyl peptides on silica gel TLC plates solvent systems were: I, ethyl acetate:methanol:water:dimethylformamide (90:5:5:0.5, by vol.); II, ethyl acetate:methanol:water:dimethylformamide (84:10:5:1, by vol.); III, ethyl acetate:propanol:methanol:water (2:2:2:1, by vol.); and IV, ethanol:water (75:25, by vol.). Protein blotting was performed by using the Fast Blot B34 semi-dry system (Biometra Göttingen, Germany) and Immobilon-P transfer membrane (Millipore) with a current density of 2.5 A/cm2 for 60 min. Washing and incubation procedures were according to standard protocols. Serum against the Red domain of LPS3 was obtained by immunization of a rabbit with LpsCRed (performed at Biogenes GmbH, Berlin). For Western blotting analysis, serum was used in 1:500 dilution. Secondary antibody was monoclonal phosphatase-conjugated anti-rabbit antibody (Sigma) at 1:20,000 dilution. Visualization of immunoreactive bands was with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate treatment at concentrations of 0.25 mg ml–1 and 0.15 mg ml–1, respectively. Enzyme Purification-All operations were performed at 4 °C. Generally, freshly harvested mycelium from 8-day-old cultures of strain Ecc93 was suspended in a 3- to 4-fold volume of buffer A (9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and passed twice through a French Press cell at 10,000 p.s.i. The homogenate was centrifuged at 20,000 × g for 15 min. The supernatant was cleared by the addition of polymin P (0.1% final concentration). After centrifugation as above, to the supernatant was added ammonium sulfate (55% final concentration) and left on ice overnight. The precipitate was collected by centrifugation at 20,000 × g for 30 min, and pellets were taken up in buffer B (9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) at final protein concentration between 15 to 20 mg ml–1. Prior to use, the protein extract was desalted into buffer B by using PD10 columns (Amersham Biosciences). For further fractionation, enzyme was concentrated by adsorption to DEAE-cellulose pre-equilibrated with buffer B and subsequent elution with 0.15 m NaCl in buffer B. 2-ml portions of such concentrated enzyme (50 mg ml–1 protein) were passed through a Superdex TM200 gel filtration column (Amersham Biosciences) previously equilibrated with buffer B at a flow rate of 1 ml min–1. 1.5-ml fractions were collected. Enzymes were localized by their ability to form thioester with radioactive substrates as [3H]dihydrolysergic acid (LPS2), [14C]alanine (LPS3), or [14C]phenylalanine (LPS1). Enzyme Assays-Enzyme thioester formations of dihydrolysergic acid or amino acids were measured as described (9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). For in vitro synthesis of ergometrine or ergopeptams, ammonium sulfate-precipitated (55% saturation) fractions desalted into buffer B on Sephadex PD10 gel filtration columns (Amersham Biosciences) were routinely used at final concentrations of 10–12 mg ml–1 protein. Standard assays contained 200 μl of extract with 2.0 μCi of [14C]alanine, 15 mm ATP, 2 mm NADPH, 20 mm MgCl2, and 0.25 mm d-lysergic acid or dihydrolysergic acid in a total volume of 200 μl (substrate mixture for each assay had been freeze-dried before and kept at –80 °C prior to use). In case of kinetic analyses, alanine concentrations were adjusted by addition of labeled or non-labeled alanine when necessary. For in vitro synthesis of ergopeptines, 200 μl (unless stated otherwise) of enzyme solution was incubated for the indicated times at 26 °C with 2 μCi of the [14C]-amino acid chosen as radiolabel, 2.5 mm of the other substrate amino acids, 15 mm ATP, 20 mm MgCl2, and 0.25 mm d-lysergic acid or dihydrolysergic acid. In case of labeling with [3H]dihydrolysergic acid, 5 μCi was used. Reactions were stopped by adding 1 ml of water and extracted twice with 2.5 ml of ethyl acetate. Combined extracts were evaporated and applied to TLC plates and developed in appropriate solvent systems (solvent system I for ergopeptams; II for ergometrine). Radioactive products were detected by radioscanning or autoradiography. For quantitation, radioactive zones were cut out from plates and counted in a liquid scintillation counter. LPS2- and LPS3-associated activities were quantitated by titration with [14C]phenylalanine or [14C]alanine, respectively, and subsequent determination of acid stable radioactivity as described (10Walzel B. Riederer B. Keller U. Chem. Biol. 1997; 4: 223-230Abstract Full Text PDF PubMed Scopus (52) Google Scholar). One unit of LPS is the amount of enzyme that binds 1 pmol of the relevant substrate in a 30-min reaction at 30 °C (in buffer B). For the analysis of enzyme-bound reaction intermediates of ergometrine synthesis, reaction incubations (in each case performed in triplicate and combined after incubation for 20 min) were precipitated with 3 ml of trichloroacetic acid (10% w/v) and left on ice for 1 h. After centrifugation and repeated washings with trichloroacetic acid and finally with EtOH, the protein pellet was dissolved in 1 ml of 1 m NaOH and left for 20 min at 37 °C. After neutralization to pH 7 with 1 m HCl, protein was removed by centrifugation, and the aqueous phase was subjected to solid-phase extraction on IFSORB as described previously (10Walzel B. Riederer B. Keller U. Chem. Biol. 1997; 4: 223-230Abstract Full Text PDF PubMed Scopus (52) Google Scholar). Radioactivity was eluted from the solid phase with methanol and after concentration subjected to TLC on silica gel using solvent systems II, III, and IV. A Cell-free System of Ergopeptam and Ergometrine Biosynthesis-To analyze the enzymatic basis in C. purpurea for the production of the two diverse d-lysergic acid alkaloid classes, a cell-free system catalyzing both ergopeptine and ergometrine synthesis was established. C. purpurea strain Ecc93 produces ergopeptines (ergocristine and ergotamine) as main components and low molecular weight ergot alkaloids, including ergometrine, as minor components of the alkaloid mixture. Ergometrine was present at 2–3% (w/w) of the total alkaloid (300 mg/liter) after 10 days of cultivation. Partially purified extracts from broken cells of strain Ecc93 showed LPS1- and LPS2-associated activity as revealed by the in vitro formation of l-ergocristam (d-lysergylvalylcyclo(phenylalanylproline) together with minor amounts of d-ergocristam (Fig. 2b) at a kcat of ∼1.2–1.4 min–1 (8–11 units/mg of protein). comparable to that of l-ergotamam synthesis catalyzed by LPSs from C. purpurea strain D1 (10Walzel B. Riederer B. Keller U. Chem. Biol. 1997; 4: 223-230Abstract Full Text PDF PubMed Scopus (52) Google Scholar). The quantitation of total LPS1-type enzyme was by measurement of capacity of enzyme fraction to catalyze formation of acid-stable [14C]phenylalanine-enzyme thioester from enzyme and [14C]phenylalanine in the presence of MgATP. Ergotamine is a minor component (15–25%) of the ergopeptine mixture elaborated by strain Ecc93. Accordingly, cell-free l-ergotamam (d-lysergylalanylcyclo(phenylalanylproline)) formation was also detectable in the protein extract, albeit at considerably lower kcat (∼0.12–0.2 min–1; not shown), which reflects either side specificity of the enzyme-catalyzing ergocristam synthesis or indicates a second LPS1 species catalyzing l-ergotamam formation present at a much lower level than that catalyzing l-ergocristam. The same enzyme extract from strain Ecc93 catalyzing ergopeptam synthesis was incubated with d-lysergic acid, MgATP, and radioactive alanine in the presence of reducing cofactors such as NADH or NADPH. Earlier work (24Agurell S. Acta Pharm. Suec. 1966; 3: 71-100PubMed Google Scholar, 25Castagnoli N. Corbett K. Chain E.B. Thomas R. Biochem. J. 1970; 117: 451-455Crossref PubMed Scopus (12) Google Scholar, 26Basmadjian G.P. Floss H.G. Gröger D. Erge D. Chem. Comm. 1969; 1: 418-419Crossref Scopus (4) Google Scholar) had suggested alanine as the precursor of the alaninol moiety of ergometrine (Fig. 1, IV), which pointed to a step requiring reducing cofactors for conversion of the carboxyl group to a hydroxymethyl group. The analysis of reaction products by TLC indeed revealed formation of a radioactive compound dependent on MgATP and NADPH (Fig. 3a). NADH was not a substrate (not shown). Because partial purified enzyme fractions from C. purpurea usually contain traces of d-lysergic acid from the fungal cells, which sticks to protein, the dependence of compound formation on d-lysergic acid initially was equivocal. However, when the same incubations contained dihydrolysergic acid instead of d-lysergic acid (i.e. in excess over endogenous d-lysergic acid traces), a compound with lower Rf value was detected, in agreement with the lower Rf value of dihydrolysergic acid in TLCs compared with d-lysergic acid (7Stadler P.A. Planta Med. 1982; 46: 131-144Crossref PubMed Scopus (22) Google Scholar). Acid hydrolysis of the two compounds yielded radioactive alaninol in each case. To confirm the lower migrating compound as dihydroergometrine, [3H]dihydrolysergic acid was incubated in the presence of non-labeled alanine (not shown). Alkaline hydrolysis of the compound in this case gave radioactive dihydrolysergic acid. Moreover, TLC cochromatography of the two sister compounds with authentic ergometrine or dihydroergometrine in solvent systems I, II, and III showed that they had the same Rf values as these standards. The enzyme activity in the extract catalyzing ergometrine synthesis was termed ergometrine synthetase. Mechanism of Ergometrine Synthesis-Reaction velocity measurements showed that the reaction catalyzed by the ergometrine synthetase proceeded for up to 60 min, with linearity for the first 10 min after which it gradually declined. Half-life time of enzyme activity on ice was 6 h. The reaction rate depended on protein concentration in a strictly linear manner over a concentration range of more than one order of magnitude (0.5–10 mg ml–1). The Km values were 123 ± 10 μm for alanine and 2–3 μm for dihydrolysergic acid and thus comparable to the values obtained previously in the case of ergotamam synthesis (9Riederer B. Han M. Keller U. J. Biol. Chem. 1996; 271: 27524-27530Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Km for NADPH was 8.7 ± 1 μm. kcat of the ergometrine producing reaction was 1.3–1.4 min–1 calculated from enzyme units obtained from measurements of [14C]alanine-enzyme thioester formation (ranging between 0.8 and 1.6 unit/mg of protein, dependent on the quality of enzyme preparation) under the assumption that the NRPS responsible for ergometrine synthesis has one binding site for alanine. Thus, ergometrine synthetase units usually were present about one order of magnitude less than LPS1 units in agreement with the fact that ergometrine is a minor component of the alkaloid mixture elaborated by C. purpurea Ecc93. To address the formation of covalently bound intermediates during ergometrine synthesis, enzyme-substrate complexes obtained from different incubations with [14C]alanine in which dihydrolysergic acid, NADPH, or ATP each that had been omitted were saponified with NaOH, and radioactive material released from the enzyme was analyzed by TLC. As was to be expected, no radioactive compounds were found when dihydrolysergic acid or ATP was omitted from incubation mixtures (Fig. 3b). By contrast, in case when NADPH was absent, a compound was split off the enzyme suggesting that NADPH plays a role in release of a covalently bound intermediate during ergometrine synthesis. Accordingly, when all substrates of the ergometrine reaction were present again no covalent intermediate was obtained, whereas ergometrine was formed (the latter not shown). This confirms the essential role of NADPH in the turnover of enzyme. The radioactive compound that accumulated in the absence of NADPH on the enzyme was identified as dihydrolysergylalanine by TLC comparison with chemically synthesized dihydrolysergylalanine in solvent systems II, III, and IV. The intermediacy of d-lysergyla