New insights into ergot alkaloid biosynthesis in Claviceps purpurea: An agroclavine synthase EasG catalyses, via a non-enzymatic adduct with reduced glutathione, the conversion of chanoclavine-I aldehyde to agroclavine

Abstract

Ergot alkaloids are indole derivatives with diverse structures and biological activities. They are produced by a wide range of fungi with Claviceps purpurea as the most important producer for medical use. Chanoclavine-I aldehyde is proposed as a branch point via festuclavine or pyroclavine to clavine-type alkaloids in Trichocomaceae and via agroclavine to ergoamides and ergopeptines in Clavicipitaceae. Here we report the conversion of chanoclavine-I aldehyde to agroclavine by EasG from Claviceps purpurea, a homologue of the festuclavine synthase FgaFS in Aspergillus fumigatus, in the presence of reduced glutathione and NADPH. EasG comprises 290 amino acids with a molecular mass of about 31.9 kDa. The soluble monomeric His(6)-EasG was purified after overproduction in E. coli by affinity chromatography and used for enzyme assays. The structure of agroclavine was unequivocally elucidated by NMR and MS analyses.

Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

“…4; Table 2). As described previously (15), the recipient strain, easA KO A. fumigatus, accumulated primarily chanoclavine and also small quantities of agroclavine (arising via a noncatalyzed isomerization of chanoclavine aldehyde [15,33]) and larger quantities of its oxidation product setoclavine (Fig. 1).…”

Genome mining reveals the presence of a conserved gene cluster for the biosynthesis of ergot alkaloid precursors in the fungal family Arthrodermataceae

“…benhamiae, Ar. otae and T. verrucosum did not indicate the existence of genes involved in the conversion of chanoclavine-I aldehyde, such as fgaOx3 and fgaFS in A. fumigatus (Wallwey et al, 2010b) or easG in C. purpurea (Matuschek et al, 2011). Furthermore, the genes in the vicinity of the five cluster genes have no significant similarity to known secondary metabolism genes.…”

“…On the other hand, the homologous genes responsible for the conversion of chanoclavine-I aldehyde, i.e. fgaOx3 and fgaFS in A. fumigatus (Wallwey et al, 2010b) or easG in C. purpurea (Matuschek et al, 2011), were not found in arthrodermataceous fungi, and nor were further genes in the specific steps of the ergot alkaloid biosynthesis.…”

“…Festuclavine then serves as a precursor for fumigaclavines A, B and C . In C. purpurea, chanoclavine-I aldehyde is converted to agroclavine by the agroclavine synthase EasG in the presence of GSH (Matuschek et al, 2011), which is then converted via elymoclavine to Dlysergic acid, the acidic component of ergoamides and ergopeptines (Haarmann et al, 2006).…”

Ergot Alkaloids

“…These include three steps of the basic pathway: apart from dmaW , the genes cloA encoding a cytochrome P450 oxidoreductase, which catalyzes the conversion of elymoclavine to paspalic acid [ 47 ], and ccsA ( easE ) encoding (a component of) the chanoclavine I synthase [ 46 ]. In addition, Matuschek et al [ 52 ] could show by heterologous expression of easG that its product (a dehydrogenase) is involved in the biosynthesis of agroclavine. Much progress has also been made in the analysis of the terminal pathway leading to ergopeptines.…”