The structural basis of psilocybin biosynthesis

Our international collaborative scientific research project between the Friedrich Schiller University in Jena and the Medal University of Innsbruck aims to reveal the secrets of magic mushrooms. Among the best-known natural products, the potent hallucinogen psilocybin represents the principal metabolite of mushrooms of the genus Psilocybe, the so-called magic mushrooms. Psilocybin containing mushrooms have been popular as a recreational drug. Importantly, from a medical perspective, psilocybin has been recognized in clinical trials as an effective antidepressant with proven pharmaceutical relevance in the treatment of existential anxiety of advanced-stage cancer patients and against depression. Despite their unique characteristics and potential use in biocatalytic natural product synthesis, the molecular structures of the fungal enzymes in the biosynthetic pathway of psilocybin are not known. Two of them, PsiD and PsiK, will likely represent entirely new unknown three- dimensional structures. The remaining three enzymes will show significant differences in numerous exposed regions and binding site details that help understanding how the fungi synthesize this potent hallucinogen from its molecular building blocks. We study the molecular structures by X-ray crystallography, where a finely focussed powerful X-ray beam is directed at a small crystal of the enzyme. Once the three-dimensional structure of the molecules is deciphered, many questions about the functional details of each synthesis step can be answered. In addition to inferring the overall three-dimensional structure, the molecular architecture of substrate channels and the catalytic sites answers questions relevant for the rational design of engineered enzymes which are used in the efficient biosynthesis of natural substances or modified therapeutic drugs. With psilocybin entering trials as a potential therapeutic agent against therapy-refractory depression and given that the details of selectivity and mechanism of key reactions are still unknown, the results will be of substantial interest to natural product chemistry researchers as well as biomedical scientists, informing about the nature and biosynthesis of a promising therapeutic agent.

Breakthrough in the study of "magic" mushrooms: the key steps in psilocybin biosynthesis Psychoactive mushrooms ("magic mushrooms") produce the hallucinogen psilocybin. In Central Europe, the inconspicuous Liberty Cap (Psilocybe semilanceata) is especially common, growing on over-fertilized meadows and along forest edges. Although psilocybin, a compound related to LSD, is subject to narcotics legislation, studies point to-though not without risk-therapeutic potential, for example in treatment-resistant depression. Its effect is based on the activation of specific serotonin (5-HT) receptors by tryptamine compounds such as psilocybin. An international research project funded by the Austrian Science Fund (FWF) and the German Research Foundation (DFG) has elucidated the genetic basis and the sequence of biosynthetic reaction steps in these fungi. Scientists at the Institute of Genetic Epidemiology have also, for the first time, described in detail the mechanism of the decisive final step: the methyltransferase PsiM from the Cuban Liberty Cap (Psilocybe cubensis) successively transfers methyl groups to the fungal substrate norbaeocystin-first forming baeocystin and, in a second step, the end product psilocybin. In the digestive tract, psilocybin is subsequently dephosphorylated by a phosphatase to psilocin, the psychoactive agent. Using high-resolution X-ray structural analyses of numerous enzyme-substrate complexes, psilocybin and its precursors were observed for the first time bound within biologically relevant enzyme complexes, and the reaction mechanism was traced at atomic detail. Critical molecular docking points that determine substrate specificity were identified, providing guidelines for the targeted biosynthesis of novel, substituted tryptamines. The results were published in the high profile journal Nature Communications. Note: Psilocybin is a regulated substance under the Narcotic Substances Act. The findings described here serve scientific understanding and do not constitute an encouragement to manufacture, possess, or use psilocybin.