In silico characterization of the psilocybin biosynthesis pathway.

Psilocybin, the psychoactive alkaloid in Psilocybe mushrooms, is synthesized from L-tryptophan by four enzymes: PsiD, PsiH, PsiK, and PsiM. Because the pathway was only recently defined, structural data for these enzymes were lacking. This work generated homology models for all four Psilocybe cubensis enzymes, refined them with secondary structure prediction, energy minimization, and molecular dynamics simulations, then validated the models by docking their natural substrates. The models produced feasible binding modes for each biotransformation step. Additional docking simulations suggest that the PsiM model can also mediate conversions leading to aeruginascin, norpsilocin, and N,N-dimethyltryptamine, indicating plausible routes for these secondary metabolites. These structural models may aid development of novel substrates, selective inhibitors, and improved in vitro psilocybin production.

Study at a glance

Abstract

Nearly all mushrooms of the Psilocybe genus contain the natural product psilocybin, which is a psychoactive alkaloid derived from l-tryptophan. Considering their use in ancient times, as well as their psychedelic properties, these mushrooms have re-emerged with psychotherapeutic potential for treating depression, which has triggered increased pharmaceutical interest. However, the psilocybin biosynthesis pathway was only recently defined and, as such, little exists in the way of structural data. Accordingly, the aim of this study was to structurally characterize this pathway by generating homology models for the four Psilocybe cubensis enzymes involved in psilocybin biosynthesis (PsiD, a decarboxylase; PsiH, a monooxygenase; PsiK, a phosphotransferase; PsiM, a methyltransferase). Following initial model generation and alignment with the identified structural templates, repeated refinement of the models was carried out using secondary structure prediction, geometry evaluation, energy minimization, and molecular dynamics simulations in water. The final models were then evaluated using molecular docking interactions with their substrates, i.e., psilocybin precursors (l-tryptophan, tryptamine, 4-hydroxytryptamine, and norbaeocystin/baeocystin), all of which generated feasible binding modes for the expected biotransformation. Further plausibility of the psilocybin → aeruginascin, 4-hydroxytryptamine → norpsilocin, and tryptamine → N,N-dimethyltryptamine conversions, all mediated by the generated model for PsiM, suggests valid routes of formation for these key secondary metabolites. The structural characterization of these enzymes and their binding modes which emerged from this study can lead to a better understanding of psilocybin synthesis, thereby paving the way for the development of novel substrates and selective inhibitors, as well as improved biotechnological manipulation and production of psilocybin in vitro.