Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase
Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase
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DOI：
10.1074/jbc.m006277200
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发表时间：
2001-01-19
影响因子：
4.8
通讯作者：
Ebel, J
中科院分区：
文献类型：
--
作者：
Latunde-Dada, AO;Cabello-Hurtado, F;Ebel, J
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。我们在这项研究中表明，CYP 71 D9 cDNA，以前分离的激发子诱导的大豆（Glycine max L.）细胞，编码具有新羟化酶活性的蛋白质。当在酵母中异源表达时，该蛋白以高亲和力（1.6至52 μ M）结合各种黄酮类化合物，并显示典型的I型吸收光谱。这些黄酮类化合物在间苯二酚和间苯三酚为基础的A-环的6位被羟基化。黄烷酮6-羟化酶（CYP 71 D9）比黄酮更有效地催化黄烷酮的转化。异黄酮几乎没有羟基化。由于大豆产生的黄酮类化合物在A环上具有6，7-二羟基取代模式，因此研究了黄酮B-羟化酶与黄酮类化合物生物合成的关系。重组8-羟基异黄烷酮合酶（CYP 93 C1 v2）有效地利用6，7，4 '-三羟基黄烷酮作为底物。为了鉴定其结构，通过酸处理将化学不稳定的反应产物转化为6，7，4 '-三羟基喹啉。两种酶的最终反应产物的结构通过MMR和质谱确认。我们的研究结果有力地支持了这样的结论，即在大豆中，在异黄烷酮形成过程中，A环的B-羟基化发生在类黄酮B环的1，2-芳基迁移之前。这是第一次从任何植物物种中鉴定类黄酮B-羟化酶cDNA。
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 muM) 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 B-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 8-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 MMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the B-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 B-hydroxylase cDNA from any plant species.