In vitro conversion of chanoclavine-I aldehyde to the stereoisomers festuclavine and pyroclavine controlled by the second reduction step

Ergot alkaloids are mycotoxins produced mainly by the genera Claviceps, Aspergillus and Penicillium. Natural and semisynthetic ergot alkaloids are used as drugs in modern medicine. Chanoclavine-I aldehyde was found to be the branch point in the biosynthesis of different ergot alkaloid types. In Claviceps purpurea, it was converted to agroclavine by agroclavine synthase EasG in the presence of reduced glutathione, while festuclavine synthase FgaFS and the old yellow enzyme FgaOx3 from Aspergillus fumigatus catalysed the formation of festuclavine by two reduction steps. In this study, pyroclavine was detected as a minor product in the incubation mixture with enzymes from A. fumigatus under optimised HPLC conditions. Furthermore, we demonstrated that the homologues of FgaFS and FgaOx3 from Penicillium commune, FgaFS(pc) and FgaOx3(pc), also catalysed the formation of pyroclavine and festuclavine, but with a significantly higher yield for pyroclavine. In addition, EasG was also shown to convert chanoclavine-I aldehyde to pyroclavine and festuclavine in the presence of the old yellow enzyme FgaOx3 or FgaOx3(pc). More importantly, the ratio of pyroclavine to festuclavine was controlled by the reduction of an imine intermediate catalysed by FgaFS, FgaFS(pc) or EasG, which was proven by using different combinations of these enzymes in the presence of FgaOx3 or FgaOx3(pc). The structure of pyroclavine was unequivocally elucidated by NMR and MS analyses.