Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Department of Agriculture-Agricultural Research Service, Wheat, Sorghum, and Forage Research Unit, Lincoln, NE 68583, USA.
Int J Mol Sci. 2024 May 22;25(11):5651. doi: 10.3390/ijms25115651.
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, , 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 -coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of -coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased 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.
查尔酮合酶 (CHS) 和查尔酮异构酶 (CHI) 催化类黄酮途径的前两个关键步骤，该途径在陆地植物的生长和繁殖中起着至关重要的作用，包括紫外线保护、色素沉着、共生固氮和抗病原体。基于来自同一单子叶植物的 CHS、CHI 和查尔酮异构酶样蛋白 (CHIL) 的 X 射线晶体结构，以及稳态动力学、光谱/热力学分析、分子间相互作用及其对每个催化步骤的影响的结果，提出了该模型。此外，还分析了 PvCHI 对柚皮素查尔酮和异甘草素的独特活性，并根据酶和两种底物的固有特性解释了对这些 I 型和 II 型底物的分级活性。与柚皮素结合的 PvCHS 结构支持非竞争性抑制。PvCHS 表现出内在的催化混杂性，这表现在除了形成柚皮素查尔酮外，还形成了 -香豆酰三乙酸内酯 (CTAL)。在 PvCHIL 的存在下，通过 PvCHS 和 PvCHI 将 -香豆酰辅酶 A 转化为柚皮素，与形成 CTAL 的减少 70%相比，增加了 ~400 倍。支持该模型，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 均以非竞争性方式与 PvCHS 相互作用，表明柚皮素对 CHS 的可能变构效应。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，表明存在一种可能的反馈机制，以最大程度地减少自发产生的不正确立体异构体。这些是来自同一物种的三体系统的首次发现，表明 CHS-CHI-CHIL 大分子组装在确定植物细胞中产生的类黄酮的数量和类型方面的重要性。
