D-lysergyl peptide synthetase from the ergot fungus Claviceps purpurea

The ergot fungus Claviceps pzrrpurea produces She medically important ergopeptines, which consist of a cyclol-structured tripeptide and D-lysergic acid linked by an amide bond, Az enzyme activity capable of nonribosomal 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) copurifyimg 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 K-m of similar to 1.4 mu M for both D-lysergic acid and its structural homolog dihydrolysergic acid, which is one to two orders of magnitude lower than the K-m values for the other amino acids involved, The K-m 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 n-lysergic acid concentration in the cell, lit 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. pupurea 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.