PlantP450Dock: an Automated Molecular Docking Pipeline of Plant Cytochrome P450s

Abstract

Cytochrome P450 enzymes (CYPs) are the primary drivers of chemical diversification in plant secondary metabolism; however, fewer than 10% of the superfamily members have been functionally characterized. Computational docking provides a scalable strategy to prioritize candidates for experimental validation, yet prevailing workflows are poorly adapted to plant P450s because AlphaFold-predicted structures lack the essential heme cofactor and conventional flexible-residue selection relies on subjective geometric cutoffs. To address these limitations, we developed an automated pipeline--PlantP450Dock--that unifies heme cofactor implantation, molecular dynamics-based conformational sampling, data-driven flexible residue selection, and semi-flexible docking within a single integrated workflow. The heme is transferred from a crystallographic template to the AlphaFold model via a local coordinate transformation algorithm, achieving a positional deviation of less than 0.2 [A] relative to the experimentally determined CYP73A33 structure (PDB: 6VBY). Subsequent 100 ns molecular dynamics simulations confirmed faithful preservation of the Fe-S coordination geometry (2.61 {+/-} 0.08 [A]) across all trajectory frames. A singular value decomposition-based heme-plane filtering strategy objectively identified distal active-site residues for flexible treatment, eliminating user-dependent subjectivity. Cross-family validation against four phylogenetically distinct P450s (CYP73, CYP711, CYP706, and CYP701) generated catalytically competent binding poses with substrate-to-heme-iron distances of 2.8-4.4 [A] without enzyme-specific parameterization. Released as an open-source tool, this pipeline furnishes the plant science community with a standardized, reproducible computational framework to accelerate functional annotation of the largely unexplored plant P450 families.