Discovery and enzymatic engineering of two non‐canonical flavonoid <scp>C3</scp> ‐hydroxylases from <i>Camptotheca acuminata</i> Decne

Hydroxylation significantly enhances the structural diversity and biological activities of flavonoids. While most characterized CYP450-type hydroxylases catalyze hydroxylation at the C2, C6, C8, C2', C3', or C5' positions of flavonoids, the canonical flavonoid C3 hydroxylase (F3H) is a 2-oxoglutarate-dependent dioxygenase. This study biochemically characterizes two non-canonical CaF3Hs, CYP71AU223 and CYP71AU224, identified from the medicinal plant Camptotheca acuminata Decne through multi-omics analysis. Quantitative expression analysis shows that CYP71AU223 is predominantly expressed in leaves and roots, whereas CYP71AU224 is primarily expressed in roots. Both CaF3Hs localize to the endoplasmic reticulum. Among them, CYP71AU223 demonstrates a higher affinity for naringenin and superior catalytic performance compared to CYP71AU224. Cross-species collinearity analysis identifies two syntenic homologs, CsCYP71A and GmCYP71A in Camellia sinensis and Glycine max, respectively, which also exhibit F3H activity. These findings indicate that non-canonical CYP450-type F3Hs are not exclusive to C. acuminata but are distributed across other flavanone-producing plants. All these newly observed CYP450-type F3Hs originate from a shared ancestral gene. Molecular docking and site-directed mutagenesis of CYP71AU223 and CYP71AU224 reveal critical residues involved in naringenin binding, including SER-131 and ASP-329 in CYP71AU223 and ARG-101 and THR-501 in CYP71AU224, which stabilize substrate orientation. Enzymatic engineering further enhances the catalytic efficiency and expands the catalytic repertoire of both enzymes. This study reports the first identification of non-canonical F3Hs across three plant species, providing molecular insights into their functions, evolutionary origins, and roles in CYP450-mediated flavonoid hydroxylation.