Functional diversification within the CYP81E subfamily involved in isoflavone biosynthesis in Glycyrrhiza glabra.

Abstract

Leguminous plants produce structurally diverse isoflavonoids, whose biological activities are influenced by regioselective hydroxylation pattern catalyzed by cytochrome P450 enzymes. Members of the CYP81E subfamily function primarily as isoflavone 2'-hydroxylases and, less frequently, isoflavone 3'-hydroxylases (I3'H), but their functional diversity remains unclear. We performed a comprehensive functional characterization of CYP81E-like genes in Glycyrrhiza glabra. Transcriptome analysis and heterologous expression in Saccharomyces cerevisiae identified CYP81E106 as an I3'H active toward multiple isoflavones, whereas CYP81E105 oxidized prenylated isoflavones and other flavonoids. MS/MS analysis indicated oxidation within the prenyl moiety rather than the flavonoid backbone. Comparative in vitro enzyme and yeast bioconversion assays revealed differences in substrate preference and conversion efficiency, highlighting the influence of cellular context on catalytic performance. These findings demonstrate the functional diversification within the G. glabra CYP81E subfamily, expanding its catalytic repertoire beyond canonical B-ring hydroxylation to prenyl-group oxidation and providing insights into isoflavonoid structural diversification in legumes.

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