Calcium activation mechanism of a noncanonical aromatic L-amino acid decarboxylase from psilocybin mushroom Psilocybe cubensis

A fungal enzyme called PcncAAAD, which decarboxylates aromatic amino acids, is activated by calcium through two metal-binding sites. The primary activation site (site A) lies between the N-terminal domain and a unique C-terminal appendage; binding calcium there stabilizes a 'lid-rim' structure that preserves the substrate-binding pocket. A secondary site (site B) within the C-terminal domain helps stabilize the enzyme's overall structure. Computer simulations and lab tests show that disrupting site A or the lid-rim severely distorts the active site and reduces or eliminates activity. Sodium does not activate the enzyme. The work clarifies how calcium activates this enzyme and may guide engineering of similar enzymes for making aromatic amino acid derivatives.

Study at a glance

Abstract

PcncAAAD is a noncanonical fungal aromatic L-amino acid decarboxylase (AAAD) featuring a unique appendage C-terminal domain (CTD) and two metal-binding sites. Unlike its mammalian and plant counterparts, PcncAAAD is activated by calcium, although the exact activation mechanism remains unclear. Here, we establish an in silico RMSD-based evaluation model through molecular dynamics simulations, validated by in vitro enzyme assays, to decipher the enzyme's calcium activation mechanism. The metal-binding site at the intra-monomer interface between the N-terminal domain and the CTD (site A) is found to play a primary role in the calcium activation of PcncAAAD, whereas the secondary site within the unique CTD (site B) contributes to the calcium-mediated stabilization of enzyme structure. Binding of calcium, but not sodium, exerts a profound influence on PcncAAAD activity by stabilizing a "lid-rim" structure underlying site A, which in turn maintains the integrity of the substrate-binding environment. In silico mutations disrupting site A or the lid-rim structure show severe structural distortion of the active site, leading to reduced or even eliminated activity as demonstrated by in vitro assays. These findings deepen our understanding of metal-activatable enzymes and hold promise for the rational design of engineered enzymes for the synthesis of aromatic amino acid derivatives.