PDF(Pubmed)
Abstract：
Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI\'s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.
摘要：
查尔酮合成酶（CHS）和查尔酮异构酶（CHI）催化类黄酮途径的前两个关键步骤，在陆地植物的生长和繁殖中起关键作用，包括紫外线防护,色素沉着,共生固氮，和病原体抗性。根据获得的CHS的X射线晶体结构，CHI,和查尔酮异构酶样蛋白（CHIL）来自同一单子叶植物，Virgatum,以及稳态动力学的结果，光谱/热力学分析，分子间相互作用，并提出了它们对每个催化步骤的影响。此外,分析了柚皮素查尔酮和异甘草素的PvCHI独特活性，用酶和两种底物的内在特征解释了观察到的I型和II型底物的分级活性。与柚皮素复合的PvCHS的结构支持非竞争性抑制。PvCHS表现出内在的催化滥交，除了柚皮素查尔酮外，还形成了对香豆酰基三乙酸内酯（CTAL）。在PvCHIL面前,通过PvCHS和PvCHI将对香豆酰基-CoA转化为柚皮素显示Vmax增加约400倍，CTAL的形成减少了70％。支持这个模型，分子对接,ITC（等温滴定量热法），和FRET（荧光共振能量转移）表明PvCHI和PvCHIL都以非竞争性方式与PvCHS相互作用，表明柚皮素对CHS的变构效应是合理的。重要的是,柚皮素的存在增加了PvCHS和PvCHIL之间的亲和力，而柚皮素查尔酮降低了亲和力，表明一个合理的反馈机制，以尽量减少自发的不正确的立体异构体。这些是来自同一物种的三体系统的第一个发现，表明CHS-CHI-CHIL的大分子组装在确定植物细胞中产生的类黄酮的量和类型中的重要性。
