Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.
Bifunctional CYP81AA proteins catalyse identical hydroxylations but alternative regioselective phenol couplings in plant xanthone biosynthesis.
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DOI：
10.1038/ncomms11472
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发表时间：
2016-05-05
影响因子：
16.6
通讯作者：
Beerhues L
中科院分区：
文献类型：
--
作者：
El-Awaad I;Bocola M;Beuerle T;Liu B;Beerhues L
氧杂蒽酮是存在于植物和微生物中的天然产物。在植物中，它们的生物合成始于2，3 ′，4，6-四羟基二苯甲酮在细胞色素P450（CYP）酶的催化下区域选择性环化为1，3，5-或1，3，7-三羟基氧杂蒽酮。本研究分离并表达了萼贯叶连翘和H.在酵母中的穿孔。微粒体催化2，4，6-三羟基二苯甲酮的3′-羟基化反应和2，3 ′，4，6-四羟基二苯甲酮的C-O苯酚偶联反应。相对于插入的3′-羟基，直向同源物Hc/HpCYP 81 AA 1通过帕拉环化形成1，3，7-三羟基氧杂蒽酮，而对位同源物HpCYP 81 AA 2将环化引导至邻位，产生异构体1，3，5-三羟基氧杂蒽酮。同源建模和相互诱变揭示了S375、L378和A483对控制HpCYP 81 AA 2的区域选择性的影响，HpCYP 81 AA 2通过六重突变转化为HpCYP 81 AA 1。然而，在HpCYP 81 AA 1的相互突变几乎不影响其区域特异性。产物对接使替代的C-O苯酚偶联反应合理化。我们的研究结果有助于了解双功能CYP的机制。 氧杂蒽酮是一种生物合成上很有吸引力的化合物。在这里，作者确定了两种细胞色素P450酶，其将二苯甲酮前体羟基化和环化为1，3，7-或1，3，5-三羟基氧杂蒽酮，并确定了决定替代区域选择性的残基。
Xanthones are natural products present in plants and microorganisms. In plants, their biosynthesis starts with regioselective cyclization of 2,3′,4,6-tetrahydroxybenzophenone to either 1,3,5- or 1,3,7-trihydroxyxanthones, catalysed by cytochrome P450 (CYP) enzymes. Here we isolate and express CYP81AA-coding sequences from Hypericum calycinum and H. perforatum in yeast. Microsomes catalyse two consecutive reactions, that is, 3′-hydroxylation of 2,4,6-trihydroxybenzophenone and C–O phenol coupling of the resulting 2,3′,4,6-tetrahydroxybenzophenone. Relative to the inserted 3′-hydroxyl, the orthologues Hc/HpCYP81AA1 cyclize via the para position to form 1,3,7-trihydroxyxanthone, whereas the paralogue HpCYP81AA2 directs cyclization to the ortho position, yielding the isomeric 1,3,5-trihydroxyxanthone. Homology modelling and reciprocal mutagenesis reveal the impact of S375, L378 and A483 on controlling the regioselectivity of HpCYP81AA2, which is converted into HpCYP81AA1 by sextuple mutation. However, the reciprocal mutations in HpCYP81AA1 barely affect its regiospecificity. Product docking rationalizes the alternative C–O phenol coupling reactions. Our results help understand the machinery of bifunctional CYPs. Xanthones are pharmacologically and biosynthetically intriguing compounds. Here, the authors identify two cytochrome P450 enzymes, which hydroxylate and cyclize the benzophenone precursor to either 1,3,7- or 1,3,5-trihydroxyxanthones, and pinpoint residues that determine the alternative regioselectivities.