Abstract
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-hydroxylase cDNA from any plant species.
Highlights
Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons
Typical enzymes belonging to the complex branch pathways for the elaboration of flavonoid skeletons are cytochrome P-450-dependent hydroxylases [3], such as flavonoid 3Ј-hydroxylase [5], flavonoid 3Ј,5Ј-hydroxylase [6], isoflavone 2Ј-hydroxylase [7], flavanone 2-hydroxylase [8], flavone synthase II [9, 10], and 2-hydroxyisoflavanone synthase (2HIS)1 [11,12,13]
Combined studies with recombinant 2-hydroxyisoflavanone synthase (2HIS; CYP93C1v2) indicated that A-ring hydroxylation occurs before the 1,2-aryl shift of the flavonoid B-ring during isoflavanone formation
Summary
Namate 4-hydroxylase, and a second one, CYP93A1 cDNA, coded for 3,9-dihydroxypterocarpan 6a-hydroxylase [17, 18]. We present the functional identification of CYP71D9 whose cDNA was previously isolated from elicitor-induced soybean cells by using the differential display method [17]. By employing heterologous expression the CYP71D9 cDNA was demonstrated to encode a protein capable of catalyzing the hydroxylation of ring A of flavonoid substrates. Combined studies with recombinant 2-hydroxyisoflavanone synthase (2HIS; CYP93C1v2) indicated that A-ring hydroxylation occurs before the 1,2-aryl shift of the flavonoid B-ring during isoflavanone formation
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