D-Lysergic acid activation and cell-free synthesis of D-lysergyl peptides in an enzyme fraction from the ergot fungus Claviceps purpurea

Abstract

The D-lysergic acid activating enzyme from the ergot fungus Claviceps purpurea was purified to near homogeneity. It has a native Mr of about 245000 and in its denatured form is a single polypeptide chain of Mr 62000. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on D-lysergic acid and, though much less, that dependent of dihydrolysergic acid. Western blot analysis of SDS electropherograms of crude protein extracts from C. purpurea using monospecific antibodies directed against the D-lysergic acid activating enzyme revealed the immunostaining of one particular band which was identical with that of the D-lysergic acid activating enzyme. No significant immunoreactive band with higher molecular weight was seen, which precludes the possibility that the enzyme had arisen from the proteolysis of a high molecular weight ergot peptide synthetase. An ammonium sulfate fractionated enzyme fraction was prepared from C. purpurea strain Cl that catalyzed the incorporation of D-lysergic acid into two peptides which besides D-lysergic acid contained alanine, phenylalanine, and proline. Dihydrolysergic acid was efficiently incorporated into the corresponding dihydrolysergic acid containing analogues of the two compounds. Radiochemical analysis and degradation studies suggest that the two D-lysergic acid containing peptides most probably are N-[N-(D-lysergyl)-L-alanyl]-L-phenylalanyl-L-proline lactam and N-[N-(D-lysergyl)-L-alanyl]-L-phenylalanyl-D-proline lactam, respectively. N-[N-(D-Lysergyl)-L-alanyl]-L-phenylalanyl-L-proline lactam is considered to be the immediate precursor of ergotamine. These data support the idea that free D-lysergic acid is a free intermediate in the biosynthesis of ergopeptines. The role of the D-lysergic acid activating enzyme is discussed.