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 245,000 and in its denatured form is a single polypeptide chain of Mr 62,000. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on D-lysergic acid and, though much less, that dependent on 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 C1 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-alanlyl]-L-phenylalanyl-L-proline lactam and N-[N-(D-lysergyl)-L-analyl]-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N -Methyl-Dimethylallyltryptophan to Chanoclavine I

“…The ku70-deficient strain derived from Claviceps purpurea strain P1 (ATCC 20102 [8,19), which produces mainly ergotamine with small amounts of ergocryptine, was described earlier (5), as were the standard media and culture conditions (19). For alkaloid production, the fungus was cultivated in T25N medium with low (0.5 g/liter KH 2 PO 4 ) and high (2.0 g/liter KH 2 PO 4 ) levels of phosphates.…”

Comparison of Ergot Alkaloid Biosynthesis Gene Clusters in Claviceps Species Indicates Loss of Late Pathway Steps in Evolution of C. fusiformis

“…C. purpurea strain P1 (ϭ ATCC 20102), which produces mainly ergotamine along with low levels of ergocryptine, was described previously (10,23), as were the standard media and culture conditions (23). C. fusiformis strain SD58 (ϭ ATCC 26245), which produces mainly agroclavine and elymoclavine, and its culture growth conditions have been described previously (21).…”

D-Lysergyl Peptide Synthetase from the Ergot Fungus Claviceps purpurea

“…Inoculum medium and medium T25 were used for alkaloid fermentations (14,15). In some occasions a modified Vogel's medium was used (12,16 [9,10-3 H]-9,10-Dihydrolysergic acid was prepared from [9,10-3 H]-9,10-dihydroergocryptine as described previously (9). Dihydrolysergic acid was kindly supplied by Dr. J.-J.…”

“…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).…”

“…They are synthesized from free D-lysergic acid, L-alanine, L-phenylalanine and L-proline with consumption of ATP (9). The formation of the (L,L,L) compound has not been detected in in vivo conditions as yet, because it appears to be converted rapidly to the corresponding ergopeptine or ergopeptam.…”