Combinatorial assembly of simple and complex D-lysergic acid alkaloid peptide classes in the ergot fungus Claviceps purpurea

Abstract

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.

FIGURE 1.

Structures of d-lysergic acid peptide alkaloids. I, d-lysergic acid; II, general structure of ergopeptines; III, general structure of ergopeptams; and IV, structure of ergometrine (synonymous with ergonovine and ergobasine).

FIGURE 2.

a, NRPS assembly line of the l-ergopeptams (ergopeptines). Ergopeptams have the general structure given in the structural formula above. In ergotamam, R₁ is methyl and R₂ is benzyl; in ergocristam R₁ is isopropyl and R₂ is benzyl; in ergocryptam R₁ is isopropyl and R₂ is isobutyl. Amino acid positions 1, 2, and 3 in the ergopeptam structure correspond to modules A1TC1 (M1), A2TC2 (M2), and A3TC3 (M3) of LPS1. The free-standing d-lysergic acid module is LPS2. b, ergocristam synthesis catalyzed by LPS1/LPS2 in crude extracts from C. purpurea strain Ecc93. The lanes show TLC separation on silica gel of extracts from reaction mixtures containing d-lysergic acid, [U-¹⁴C]valine, phenylalanine, and proline, with and without MgATP. Authenticity of reaction products was checked by radiochemical analysis as described previously (9). The solvent system was solvent system I.

FIGURE 3.

a, ergometrine synthesis catalyzed by ergometrine synthetase in crude extract from C. purpurea strain Ecc93. The lanes show TLC separation on silica gel of extracts from reaction mixtures containing d-lysergic acid (or dihydrolysergic acid), [U-¹⁴C]alanine, MgATP, and NADPH. Each lane represents an experiment with the addition or omission of the indicated substrate. The solvent system was solvent system II. b, analysis of enzyme-bound reaction intermediates of ergometrine synthesis bound to ergometrine synthetase. The lanes show TLC separation on a silica gel of radioactive material that was split off ergometrine synthetase when incubated with different reaction mixtures. These mixtures contained dihydrolysergic acid, alanine, MgATP, and NADPH. Each lane represents an experiment with the addition or omission of the indicated substrate. [U-¹⁴C]Alanine was used as radiolabel in each experiment. The solvent system was solvent system III. Note: covalently bound reaction product is only visible in the case of NADPH. Authentic dihydrolysergylalanine (10) was used as the chromatographic standard. The shadow on the autoradiogram is from a radioactive contamination in the screen.

FIGURE 4.

Fractionation and complementation analysis of LPS enzymes of C. purpurea strain Ecc93 by gel filtration on Superdex TM200. Ammonium sulfate- and DEAE-cellulose-fractionated enzyme extract was applied onto a Superdex TM200 (Amersham Biosciences) column, and fractions were assayed for enzyme-thioester formation of LPS1 (with [U-¹⁴C]phenylalanine (•)), LPS2 (with [³H]dihydrolysergic acid (▪)), and ergometrine synthetase (with [U-¹⁴C]alanine (▴)). Each peak was tested for complementation with the others by incubating 150 μl of each peak fractions of LPS1, LPS2, and ergometrine synthetase in standard conditions, except that the volume was 350 μl instead of 200 μl. Label for ergotamam synthesis was [U-¹⁴C]phenylalanine, for ergometrine synthesis [U-¹⁴C]alanine. All reaction products were chromatographed on silica gel plates in solvent system II (upper panels). Note, that in the conditions used only d-ergotamam is formed due to the presence of 6 mm dithiocrythritol in the flow buffer, which was not desalted prior to incubation.

FIGURE 5.

Identification of LPS3 from C. purpurea Ecc93 and proof of its structure. a, structure derived from the lpsC sequence in the ergot alkaloid biosynthesis gene cluster of C. purpurea strain P 1 (16) (derivative of C. purpurea ATCC 20102). A, adenylation; T, thiolation; C, condensation; R, reductase; calculated M_r: 178,200. The proposed reaction mechanism was in conjunction with LPS2. b, immunoblot analysis of ergometrine synthetase fractions of the gel filtration separation in Fig. 4 with antibody raised against the Red-domain of LPS3 encoded by the lpsC sequence of Ecc93. The analysis shows that LPS3 is present only in those fractions catalyzing alanine thioester formation and complementing with LPS2 in the biosynthesis of ergometrine (shown below). c, corrected domain arrangement of LPS3 showing the C0-domain, which contributes 19 kDa to the originally calculated molecular mass of the enzyme in accordance with the experimentally determined value.

FIGURE 6.

Alignment of N-terminal regions of LPS enzymes. The beginning of the corresponding A-domains is denoted by the conserved WN at the end of each sequence. C5 and C6 denote the signature consensus of C5- and C6-motifs of C-domains of NRPSs. The helical region α7/8 is defined by comparison with the VibH sequence and by modeling analysis using the PHYRE fold recognition server (40). The arrows denote the locations of the introns in the C0- and the C2-domains in the alignment and in the domain organizations of LPS enzymes given below. Intron boundaries are boxed.

FIGURE 7.

Phylogenetic analysis of AT-didomains and C-domains of the various LPSs from ergot fungi. AT-didomains: Analysis was done using ClustalW using sequences of AT-didomains of LPS2 and from several other fungal NRPS and of an NRPS (CTS) from C. purpurea strain D1 (ATCC 20102) unrelated to d-lysergic acid alkaloid biosynthesis. The shading under the clades contains AT-didomains from the first modules (M1) and the third modules (M3) (clade I), and the second modules (M2) of LPS enzymes (clade II). The other AT-didomains from other fungi or from C. purpurea (CTS) are unrelated to alkaloid peptide synthesis. The accession numbers are as follows: LPS1 (C. purpurea) (CAB39315); LPS4 (C. purpurea) (CAI59268); LPSA (N. lolii) (AAL26315); LPS2 (C. purpurea) (CAD28788); LPSB (N. lolii) (ABM91454); LPS3 (C. purpurea) (CAI59267); HC-toxin synthetase (C. carboneum) (Q01886); CTS (C. purpurea) (ABR23346); AAX63399 (Hypocrea virens) (AAX63399); aminoadipylcysteinylvaline synthetase (Penicillim chrysogenum) (P26046); and VibH (Vibrio cholerae) (AAD48879). C-domains: phylogenetic tree of all C-domains of LPS enzymes along with VibH-C-terminal half domain. The phylogenetic trees were generated with ClustalW using the amino acid sequences of C-terminal halves of the regular C-domains and of C0-domains (for detail see supplemental material). The phylogenetic tree was essentially the same when full-length C-domain sequences were used.