Flavonoid 6-Hydroxylase from Soybean (Glycine maxL.), a Novel Plant P-450 Monooxygenase

作者：A. O. Latunde‐Dada, Francisco Cabello‐Hurtado, Nikola Czittrich, Luc Didierjean, Christel R Schopfer, Norbert Hertkorn, Danièle Werck‐Reichhart, Jürgen Ebel · 发表于：Journal of Biological Chemistry · 年份：2001 · DOI：10.1074/jbc.m006277200 · 被引用次数：147 · 研究领域：Plant Gene Expression Analysis、Phytoestrogen effects and research、Phytochemicals and Antioxidant Activities

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification 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 affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically 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 NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydrox...