Latunde-Dada A O, Cabello-Hurtado F, Czittrich N, Didierjean L, Schopfer C, Hertkorn N, Werck-Reichhart D, Ebel J
Botanisches Institut der Universität, Menzinger Strasse 67, D-80638 München, Germany.
J Biol Chem. 2001 Jan 19;276(3):1688-95. doi: 10.1074/jbc.M006277200. Epub 2000 Oct 10.
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.
细胞色素P-450依赖性羟化酶是修饰基本类黄酮骨架的典型酶。我们在本研究中表明，先前从激发子诱导的大豆（Glycine max L.）细胞中分离出的CYP71D9 cDNA编码一种具有新型羟化酶活性的蛋白质。当在酵母中异源表达时，该蛋白质以高亲和力（1.6至52微摩尔）结合各种类黄酮，并显示出典型的I型吸收光谱。这些类黄酮在基于间苯二酚和间苯三酚的A环的6位被羟基化。类黄酮6-羟化酶（CYP71D9）催化黄烷酮的转化比黄酮更有效。异黄酮几乎不被羟基化。由于大豆产生在A环上具有6,7-二羟基取代模式的异黄酮成分，因此研究了类黄酮6-羟化酶与异黄酮生物合成的生物合成关系。重组2-羟基异黄酮合酶（CYP93C1v2）有效地使用6,7,4'-三羟基黄烷酮作为底物。为了进行结构鉴定，通过酸处理将化学不稳定的反应产物转化为6,7,4'-三羟基异黄酮。两种酶的最终反应产物的结构通过核磁共振和质谱法得到证实。我们的结果有力地支持了这样的结论，即在大豆中，A环的6-羟基化发生在异黄酮形成过程中类黄酮B环的1,2-芳基迁移之前。这是首次从任何植物物种中鉴定出类黄酮6-羟化酶cDNA。
