Abstract
The ergot fungus Claviceps purpurea produces the medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond. An enzyme activity capable of non-ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea strain D1. Analysis of radioactively labeled enzyme-substrate complexes revealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the amino acid activation domains for alanine, phenylalanine, and proline. The activation of D-lysergic acid is catalyzed by a 140-kDa peptide synthetase (LPS 2) copurifying with LPS 1. LPS 1 and LPS 2 contain 4'-phosphopantetheine and bind their substrates covalently by thioester linkage. Kinetic analysis of the synthesis reaction revealed a Km of approximately 1.4 microM for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the Km values for the other amino acids involved. The Km values for the amino acids reflect their relative concentrations in the cellular pool of C. purpurea. This may indicate that in in vivo conditions D-lysergyl peptide formation is limited by the D-lysergic acid concentration in the cell. In vitro, the multienzyme preparation catalyzes the formation of several different D-lysergyl peptide lactams according to the amino acids supplied. Specific antiserum was used to detect LPS 1 in various C. purpurea strains. In C. purpurea wild type, the enzyme was expressed at all stages of cultivation and in different media, suggesting that it is produced constitutively.
Highlights
The ergot fungi growing on their host plants develop characteristic sclerotia containing ergot alkaloids
Purification of D-Lysergyl Peptide-synthesizing Enzyme—C. purpurea strain D1 produces ergotamine and minor amounts of Leu-ergokryptine
The results shown here suggest that lysergyl peptide synthetase 1 (LPS 1) and LPS 2 can synthesize various different Dlysergyl peptides and that the diversity of structures elaborated by the various C. purpurea strains, at least in part, reflects the different actual concentrations of substrate amino acids in their free cellular pools
Summary
The ergot fungi growing on their host plants develop characteristic sclerotia containing ergot alkaloids. Called ergopeptines, are produced by Claviceps purpurea and consist of cyclol-structured tripeptides attached by amide linkage to D-lysergic acid (Fig. 1a). It has been postulated that the cyclol in the ergopeptines arises by introduction of a hydroxyl group to the ␣-C of the amino acid I of a putative D-lysergyl (L,L,L)tripeptide lactam [6, 7]. We have previously reported a cell-free system capable of forming the novel D-lysergyl peptides D-lysergyl-L-alanyl-L-phenylalanyl-L-proline lactam and the corresponding D-proline containing stereoisomer [9]. They are synthesized from free D-lysergic acid, L-alanine, L-phenylalanine and L-proline with consumption of ATP [9]. This study is dedicated to the memory of the late Dr Hans Kobel (deceased 1992), a pioneer in the field of peptide alkaloid research and of microbial secondary metabolism
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