Claviceps purpurea produces the pharmacological important ergopeptines, a class of cyclol-structured alkaloid peptides containing D-lysergic acid. These compounds are assembled from D-lysergic acid and three different amino acids by the nonribosomal peptide synthetase enzymes LPS1 and LPS2. Cloning of alkaloid biosynthesis genes from C. purpurea has revealed a gene cluster including two NRPS genes, cpps 1 and cpps 2. Protein sequence data had assigned earlier cpps1 to encode the trimodular LPS1 assembling the tripeptide portion of ergopeptines. Here, we show by transcriptional analysis, targeted inactivation, analysis of disruption mutants, and heterologous expression that cpps 2 encodes the monomodular LPS2 responsible for D-lysergic acid activation and incorporation into the ergopeptine backbone. The presence of two distinct NRPS subunits catalyzing formation of ergot peptides is the first example of a fungal NRPS system consisting of different NRPS subunits.

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 (1). 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 (2, 3). The natural D-lysergic acid amides are produced by a wide variety of ascomycete fungi, mostly belonging to the family Clavicipitaceae (4). 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 (5). 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 (6).In the ergopeptines (see Fig. 1, II) D-lysergic acid is amidated with bicyclic tripeptide chains. The first tw...

Clavines and D-lysergic acid-derived alkaloid amides and alkaloid peptides are two different families of compounds that have the indole-derived tetracyclic metergoline ring system in common. Previous work has shown that D-lysergic acid is biosynthetically derived from clavine alkaloids. Recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from the D-lysergic acid peptide (ergopeptines)-producing Claviceps purpurea, has shown that it most probably contains all genes necessary for D-lysergic acid synthesis as well as those that encode the assembly of D-lysergic acid peptides, such as ergotamine. To address the role of the oxygenase genes of alkaloid-gene clusters, the only cytochrome P450 monooxygenase gene of this cluster was inactivated by disruption. The resultant mutant accumulated agroclavine, elymoclavine, and chanoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D-lysergic acid restored ergopeptine synthesis; this suggests a block in the conversion of elymoclavine to D-lysergic acid. The gene was designated cloA (for encoding a clavine oxidase, CLOA). Retransformation of the mutant with the intact cloA gene also restored ergopeptine synthesis. These data show that CLOA catalyses the conversion of clavines to D-lysergic acid, it acts as a critical enzyme in the ergot alkaloid gene cluster, and bridges the biosynthesis of the two different families of alkaloids.

Subtle restraint: Abyssomicin C and atrop‐abyssomicin C are polyketide‐type antibiotics produced by the marine actinomycete of the genus Verrucosispora. Investigations of the functional pathway show that a subunit of 4‐amino‐4‐deoxychorismate synthase from Bacillus subtilis is irreversibly inhibited through covalent binding to the side chain of Cys 263, undergoing a rearrangement to a structure of the abyssomicin D type (see scheme).

Subtile Hemmung: Abyssomicin C und atrop‐Abyssomicin C sind Antibiotika vom Polyketid‐Typ, die beide von marinen Actinomyceten der Gattung Verrucosispora produziert werden. Studien zum Wirkmechanismus belegen, dass eine Untereinheit der 4‐Amino‐4‐desoxychorismat‐Synthase aus Bacillus subtilis durch kovalente Bindung von atrop‐Abyssomicin C an die Seitenkette Cys 263 irreversibel inhibiert wird, wobei eine Umlagerung zu einer Struktur ähnlich der von Abyssomicin D stattfindet (siehe Schema).