植物叶黄质合成中细胞色素P450酶的结构与机理研究

Lutein, the most abundant carotenoids in chloroplasts, plays an important role in photoprotection by acting as a quencher in the light-harvesting complexes. Lutein is synthesized from α-carotene by two hydroxylation steps: hydroxylation of the β-ring by CYP97A3/LUT5, and then ε-ring by CYP97C1/LUT1. Both enzymes have an N-terminal transmembrane region, a characteristic of most eukaryotic cytochrome P450 enzymes. The catalytic mechanism of this huge family of heme-containing enzymes is the frontier area of biochemistry. However, structural and mechanistic studies on plant cytochrome P450 enzymes are rarely reported. The electron donor for CYP97A3/LUT5 and CYP97C1/LUT1 remains unknown and little is understood about the electron transfer pathway during catalysis. Here we propose to investigate the structure and mechanistics of the two cytochrome P450 enzymes in an attempt to further the study of plant cytochrome P450 systems. In combination with studies on photosynthesis and plant physiology, we will address the fundamental biochemical issues such as their kinetic characteristics, identity of the electron donor(s), electron transfer pathway, recognition of the substrate, and the catalytic procedure.

叶黄质是叶绿体里含量最丰富的类胡萝卜素，其主要功能是作为捕光复合物中的淬灭剂，在光保护过程中发挥着重要的作用。叶黄质由α-胡萝卜素经两步加氧反应生成：CYP97A3/LUT5催化的β环羟化和CYP97C1/LUT1催化的ε环羟化。与许多已知的真核P450酶类似，这两个P450酶也是N端穿膜、固定在膜上的蛋白。P450酶的催化机理研究一直是生物化学的前沿领域，但目前，对植物P450酶的催化机理研究非常少。CYP97A3/LUT5和CYP97C1/LUT1定位于叶绿体中，为这两个P450酶提供电子的供体尚未确定，催化过程中电子传递的方式仍有待阐明。本项目拟开展对植物叶黄质合成途径中的P450酶的结构和机理的生物学研究。结合植物光合与生理学，项目申请人将探讨叶黄质合成中的P450酶的酶学特征、电子供体和电子传递方式、底物识别以及催化过程，促进对植物P450酶体系的生物化学研究。

叶黄质由α-胡萝卜素通过两步羟化反应生成，细胞色素P450单加氧酶CYP97A催化底物α-胡萝卜素一端β环上C3的羟化反应，CYP97C则催化另一端ε环上C3的羟化反应。底物的β环与ε环由于烯键位置不同而产生细微的结构差异，CYP97A和CYP97C能够区分两者并完成pro-3R的特异性羟基化。项目组解析了模式植物拟南芥的CYP97A3和CYP97C1的晶体结构。CYP97A3结合视黄醛的复合物结构展示了pro-3R专一性羟基化的分子基础，CYP97C1结合去垢剂分子的复合物结构确定了底物通道的位置。项目组建立了体外CYP97A3的酶活体系，确定了电子供体由[2Fe-2S]铁氧还蛋白与铁氧还蛋白-NADP+还原酶构成。本项研究首次报道了叶绿体P450酶的结构，揭示了植物合成叶黄质最后两步反应的分子机理。