Characterization of the Gateway Decarboxylase for Psilocybin Biosynthesis

The l‐tryptophan decarboxylase PsiD catalyzes the initial step of the metabolic cascade to psilocybin, the major indoleethylamine natural product of the “magic” mushrooms and a candidate drug against major depressive disorder. Unlike numerous pyridoxal phosphate (PLP)‐dependent decarboxylases for natural product biosyntheses, PsiD is PLP‐independent and resembles type II phosphatidylserine decarboxylases. Here, we report on the in vitro biochemical characterization of Psilocybe cubensis PsiD along with in silico modeling of the PsiD structure. A non‐canonical serine protease triad for autocatalytic cleavage of the pro‐protein was predicted and experimentally verified by site‐directed mutagenesis. How the magic begins: The l‐tryptophan decarboxylase PsiD initiates the biosynthesis of the psychotropic natural product psilocybin in Psilocybe mushrooms. Its in vitro biochemical characterization and in silico modeling of its structure identified a non‐canonical serine protease triad for autocatalytic cleavage of the proenzyme. PsiD's substrate flexibility makes it a versatile catalyst for biotechnological applications.