Calcium Activation Mechanism of a Noncanonical Aromatic L-Amino Acid Decarboxylase from Psilocybin Mushroom

PcncAAAD is a calcium-activatable noncanonical aromatic L-amino acid decarboxylase (AAAD) featuring a unique appendage C-terminal domain (CTD) and two metal-binding sites. In this study, 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. Between the two metal-binding sites, the site at the N-terminal domain/CTD interface (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. Collectively, our computational and experimental analyses pinpoint the molecular mechanism underlying the noncanonical activation of PcncAAAD by calcium. 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.