Functional Characterization of Isoflavonoid Synthase CYP736D1 Mediating Oxidative Aryl Migration in a Non-leguminous Plant, Iris germanica (Iridaceae)

Abstract Isoflavonoids are a specialized subclass of flavonoids predominantly distributed in the Fabaceae, where they fulfill essential ecophysiological roles as antimicrobial phytoalexins and symbiotic signal molecules. During their biosynthesis, 2-hydroxyisoflavanone synthase [also called isoflavonoid synthase (IFS)], a cytochrome P450 (P450) of the CYP93C subfamily, catalyzes the oxidative aryl migration of (2S)-flavanones. While various species in the genus Iris (Iridaceae, monocots) are also prolific isoflavonoid producers, the biosynthetic mechanisms in these non-leguminous plants have remained elusive. To identify the Iris IFS, we utilized an established adventitious-root culture system for Iris germanica and employed functional expression–fractionation screening of its cDNA library in yeast cells. This sequence-independent strategy successfully identified CYP736D1 as the enzyme mediating the characteristic skeletal rearrangement of naringenin to form 2-hydroxyisoflavanone. CYP736D1 showed higher in vitro and in vivo catalytic activities than CYP93C2, owing to its higher substrate affinity, kinetic efficiency, and protein stability. The IFS activity was highly specific to naringenin; reactions with other flavanones (liquiritigenin, hesperetin, and pinocembrin) primarily yielded corresponding flavone or 3-hydroxyflavanones. The reaction mechanism of CYP736D1 is considered to be essentially identical to that of CYP93C, but the amino acid residues of CYP93C critical for the IFS activity are not conserved in CYP736D1. Thus, IFS proteins in the Fabaceae and Iridaceae likely evolved from different ancestral P450s and independently acquired the ability to construct the isoflavonoid skeleton through parallel evolution. Bioconversion and transgenic experiments demonstrated that CYP736D1 is a promising tool for biotechnological isoflavonoid production in heterologous systems.