Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450monooxygenase

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modific ation of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA , previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affini ty (1.6 to 52 muM) and showed typical type I absorption spectra. These flav onoids were hydroxylated at position 6 of both resorcinol- and phloroglucin ol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversio n of flavanones more efficiently than flavones. Isoflavones were hardly hyd roxylated. As soybean produces isoflavonoid constituents possessing 6,7-dih ydroxy substitution patterns on ring A, the biosynthetic relationship of fl avonoid B-hydroxylase to isoflavonoid biosynthesis was investigated. Recomb inant 8-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-tr ihydroxyflavanone as substrate. For its structural identification, the chem ically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by MMR and mass spectrometry. Our results strongly s upport the conclusion that, in soybean, the B-hydroxylation of the A-ring o ccurs before the 1,2-aryl migration of the flavonoid B-ring during isoflava none formation. This is the first identification of a flavonoid B-hydroxyla se cDNA from any plant species.