Abstract

(-)-Epigallocatechin has multiple bioactivities. ANR catalyzes its biosynthesis, with unclear structure and mechanism restricting enzyme modification. Here, we first screened ANRs from 21 plant species and identified Torreya grandis ANR (TgANR) as the most efficient candidate based on molecular docking and in vitro assays. Through integrated alanine scanning and computational prediction, we constructed a combinatorial mutant N15S/F106E/P182S, which exhibited a 3.88-fold higher EGC synthesis activity than the wild-type enzyme. The interaction mechanism between TgANR and the substrate delphinidin as well as the structure-activity relationship in the catalytic production of EGC were studied on the basis of protein engineering modification, thus clarifying the catalytic mechanism of TgANR. Furthermore, transient overexpression of the mutant gene in T. grandis kernels significantly increased EGC accumulation. This study not only elucidates the structure activity relationship of TgANR but also provides an efficient enzyme variant and a protein engineering framework for boosting EGC biosynthesis in plants.