结构引导鉴定 ZmGA20ox 催化决定簇，实现玉米赤霉素工程化 CRISPR 靶点的理性选择

Gibberellin 20-oxidase is a key enzyme controlling gibberellin biosynthesis and represents an attractive target for engineering plant architecture and drought tolerance in maize. In this study, we developed a structure-guided computational workflow to investigate functionally important residues in maize gibberellin 20-oxidase 3 (ZmGA20ox3), integrating AI-based structural prediction, molecular docking, molecular dynamics simulations, and in vitroCRISPR/Cas9 guide RNA cleavage analysis. Structural analysis revealed the conserved Fe²+-binding catalytic triad (His147-Asp149-His166) surrounded by an aromatic substrate-recognition pocket comprising Phe151, Trp157, and Phe163. Docking analyses showed that native gibberellin substrates and the co-substrate 2-oxoglutarate occupied the predicted catalytic cavity, while prohexadione exhibited the most favourable predicted docking score among the tested ligands. Computational mutagenesis revealed generally less favourable predicted docking scores for the catalytic-site substitutions H147A, D149A, and H166A, whereas substitutions at aromatic substrate-recognition residues produced ligand-dependent effects. Subsequent 100 ns molecular dynamics simulations revealed distinct ligand-dependent conformational behaviours, with H147A affecting the stability of the predicted catalytic environment, whereas W157A primarily influenced ligand accommodation within the substrate-binding pocket. MM/GBSA analyses further indicated that favourable predicted ligand interactions do not necessarily imply a catalytically competent active-site configuration, highlighting the importance of productive active-site organization. Finally, in vitro CRISPR/Cas9 cleavage assays demonstrated sequence-specific cleavage of guide RNAs targeting the ZmGA20ox3 regions containing H147 and W157, supporting the feasibility of targeting these sites in subsequent genome-editing experiments. Collectively, this study provides structure-guided computational insights into the potential roles of key residues in ZmGA20ox3 and establishes a predictive framework for prioritizing candidate residues for future biochemical and genome-editing validation.