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-ly sergic acid linked by an amide bond, Az enzyme activity capable of non ribosomal synthesis of D-lysergyl-L-alanyl-L-phenylalanyl-L-proline la ctam, the non-cyclol precursor of the ergopeptine ergotamine, has been purified about 18-fold from the ergotamine-producing C. purpurea stra in D1. Analysis of radioactively labeled enzyme-substrate complexes re vealed a 370-kDa lysergyl peptide synthetase 1 (LPS 1) carrying the am ino acid activation domains for alanine, phenylalanine, and proline. T he activation of D-lysergic acid is catalyzed by a 140-kDa peptide syn thetase (LPS 2) copurifyimg with LPS 1, LPS 1 and LPS 2 contain 4'-pho sphopantetheine and bind their substrates covalently by thioester link age, Kinetic analysis of the synthesis reaction revealed a K-m of simi lar to 1.4 mu M for both D-lysergic acid and its structural homolog di hydrolysergic 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-l ysergyl peptide formation is limited by the n-lysergic acid concentrat ion 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 w as expressed at all. stages of cultivation and in different media, sug gesting that it is produced constitutively.