Towards an Alternate Psilocybin Biosynthesis Pathway Starting with Methylation of 4-Hydroxytryptophan

Psilocybin, the psychedelic compound found in a variety of bioactive mushroom species, has been used in clinical trials to study its efficacy towards the treatment of mental health disorders such as depression, anxiety, and post-traumatic stress disorder. The in vivo production of psilocybin follows a 4-hydroxyindole supplemented, three-step pathway that includes the enzymes psiD, psiK, and psiM. PsiM is a methyltransferase responsible for the conversion of norbaeocystin to baeocystin, and baeocystin to psilocybin by means of the methyl donor S-adenosyl-L-methionine (SAM). However, another SAM dependent N-methyltransferase derived from the Psilocybe serbica mushroom, TrpM, could lead to an alternative psilocybin production pathway that excludes the norbaeocystin methyltransferase, psiM. This novel pathway begins with the iterative methylation of L-tryptophan and results in mono-, di- and trimethylated products that could provide a possible substrate to psiD and psiK. In order to test this, the in vivo activity of TrpM needed to be examined. An E. coli vector containing the TrpM gene was put through a well plate assay resulting in the expected mono-, di-, and trimethylated products. Using 4-hydroxyindole as a supplement, TrpM was also able to carry out its processes on 4-hydroxytryptophan. This resulted in mono- and dimethylated products. This proposed alternative pathway begins with TrpM due to previously published findings that TrpM and psiM are not interchangeable; this is due to the different substrates that they accept. Once TrpM methylates 4-hydroxytryptophan, psiD can decarboxylate N,N-dimethyl-4-hydroxytryptophan, forming psilocin, and psiK can then phosphorylate to obtain the final product of psilocybin. Our efforts to evaluate this alternate biosynthesis pathway towards psilocybin will be presented along with analytical LCMS analysis demonstrating the first report of enzymatic N-methylation of 4-hydroxytryptophan in vivo.