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.
Data availability
The source data supporting the findings from this study are available via the CUHK Research Data Repository at https://doi.org/10.48668/WDGXNE53. All parameters, structures, and MD simulation inputs/outputs for each system investigated in this study are publicly accessible through Figshare at https://doi.org/10.6084/m9.figshare.3095085854. The Uncropped (or less cropped with all lanes remained), unedited gel images are provided in Supplementary Fig. 21. The raw mass spec data and associated metadata are publicly accessible through Zenodo repository at https://doi.org/10.5281/zenodo.1848776255.
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Acknowledgements
This work has been supported by project 14305121 from the Hong Kong Research Grants Council as well as direct grants from the Chinese University of Hong Kong (Y. W.) and the Keck Foundation (J.K.W.). We acknowledge Lauren Piasecki for running the SDS-PAGE gel in Supplementary Fig. 19b.
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T.L. and E.R. contributed equally to this work. Y.W. conceived the idea. T.L. designed the mutants, performed the classical MD simulations, analyzed the data, and drafted the manuscript. E.R. cloned, expressed, and purified the proteins, and performed in vitro enzyme reactions, kinetics calculations, and ThermoFluor assays. T.L. and Z.W. performed the Drude simulation. M.T. discussed the results. Y.W. and J.W. provided supervision and edited the final version of the manuscript. All authors participated in the manuscript writing and approved the final version of the manuscript.
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J.K.W. is a member of the Scientific Advisory Board and a shareholder of DoubleRainbow Biosciences, Galixir, and Inari Agriculture, which develop biotechnologies related to natural products, drug discovery and agriculture. All other authors have no competing interests.
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Communications Biology thanks Mariarita Bertoldi, Feng Qi and the other anonymous reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Laura Rodríguez Pérez.
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Li, T., Reynolds, E.E., Wang, Z. et al. Calcium activation mechanism of a noncanonical aromatic L-amino acid decarboxylase from psilocybin mushroom Psilocybe cubensis. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09756-y
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DOI: https://doi.org/10.1038/s42003-026-09756-y
