Monoamine Biosynthesis via a Noncanonical Calcium-Activatable Aromatic Amino Acid Decarboxylase in Psilocybin Mushroom

A newly characterized enzyme from the psilocybin mushroom Psilocybe cubensis, called PcncAAAD, can decarboxylate several aromatic amino acids—including L-tryptophan, L-phenylalanine, and L-tyrosine—as well as chloro-tryptophan derivatives. Unlike previously known aromatic L-amino acid decarboxylases (AAADs) in mammals and plants, this enzyme belongs to a different protein family and contains a unique C-terminal double-β-barrel domain that binds calcium, which is required for its activity and thermal stability. The enzyme likely contributes to psilocybin biosynthesis and offers a new tool for engineering production of aromatic-amino-acid-derived natural products.

Study at a glance

Abstract

Aromatic l-amino acid decarboxylases (AAADs) are a phylogenetically diverse group of enzymes responsible for the decarboxylation of aromatic amino acid substrates into their corresponding aromatic arylalkylamines. AAADs have been extensively studied in mammals and plants as they catalyze the first step in the production of neurotransmitters and bioactive phytochemicals, respectively. Unlike mammals and plants, the hallucinogenic psilocybin mushroom Psilocybe cubensis reportedly employs an unrelated phosphatidylserine-decarboxylase-like enzyme to catalyze l-tryptophan decarboxylation, the first step in psilocybin biosynthesis. To explore the origin of this chemistry in psilocybin mushroom, we generated the first de novo transcriptomes of P. cubensis and investigated several putative l-tryptophan-decarboxylase-like enzymes. We report the biochemical characterization of a noncanonical AAAD from P. cubensis ( PcncAAAD) that exhibits substrate permissiveness toward l-phenylalanine, l-tyrosine, and l-tryptophan, as well as chloro-tryptophan derivatives. The crystal structure of PcncAAAD revealed the presence of a unique C-terminal appendage domain featuring a novel double-β-barrel fold. This domain is required for PcncAAAD activity and regulates catalytic rate and thermal stability through calcium binding. PcncAAAD likely plays a role in psilocybin production in P. cubensis and offers a new tool for metabolic engineering of aromatic-amino-acid-derived natural products.