An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2 ), an important intermediate produced by the enzyme EasD af or its counterpart FgaDH from chanoclavine-I (CC, 1 ). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3 ), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasD af not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. Key points • EAs possess complicated skeletons and are widely used in several clinical diseases • EasD af belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasD af for dehydrogenation was analyzed by molecular docking and site mutations