多酚氧化酶典型酚类底物特异选择性的分子机制

Targeting inhibition of polyphenol oxidase (PPO) is an effective method for browning process control of plants and agricultural by-products. Elucidating the substrate specificity mechanism of PPO on phenolic substrate compounds is the theoretical basis for targeted design of inhibitors. While there are few reports about the relative research till now. The structural analysis of the functional domain of PPO and the confirmation of the key amino acids are the bottlenecks that restrict the ascertainment of specific mechanisms. In our previous study, the structure model of lotus seed PPO has been constructed, and the binding mode between PPO and four phenolic substrates is simulated, as well as the biochemical mechanism of lotus seed PPO induced browning is discussed. On the basis above, the lotus seed and root, which with the same genus but still specific in substrates, were selected as the research object. And then the key amino acids in the catalytic domain of PPO were identified by site directed mutagenesis, the specific binding process of PPO and phenolic substrates was observed by molecular simulation, and the changes in the spatial structure of catalytic functional domains during the binding process were verified by means of bio spectroscopy; The intermediate state structure information of the enzyme substrate was observed by the stop flow technique, and to confirming the precise identification, localization and binding sites of the specific substrate on structural domain by PPO. In this way, we try to elucidate the molecular binding mechanism of PPO on the specific phenolic substrates during the browning, which may provide theoretical supports for researching and developing of novel high efficient inhibitors of agricultural by-products.

多酚氧化酶（PPO）的靶向抑制是植物性农副产品褐变控制的有效手段。明确PPO对底物特异选择性的分子机制是进行抑制剂靶向设计的基础，而此类研究目前尚鲜有报道。PPO催化功能域的结构解析及关键氨基酸的确证是制约特异性机制研究的瓶颈。申请人前期已构建了莲子PPO的结构模型，并模拟了PPO与四种酚类底物之间的结合模式，探讨了莲子PPO促褐变的生化机制。本项目拟在此基础上，选择同种属PPO结构相近但其特异性底物不同的莲子、莲藕为研究对象，通过定点突变确定PPO催化功能域的关键氨基酸，借助分子模拟技术观测PPO与酚类底物的特异性结合过程，辅以生物谱学表征手段验证结合过程中催化功能域空间构象的变化；采用停流光谱联用技术观察酶底物中间态结构信息，确证PPO精准识别、定位、结合特异性底物的位点和结构功能域。基于此，阐明PPO底物特异选择性的分子机制，为植物性农副产品褐变抑制剂的研发提供理论支撑。

多酚氧化酶（PPO）的靶向抑制是植物性农副产品褐变控制的有效手段。明确PPO对底物特异选择性的分子机制是进行抑制剂靶向设计的基础，而此类研究目前尚鲜有报道。PPO催化功能域的结构解析及关键氨基酸的确证是制约特异性机制研究的瓶颈。本项目针对植物性农副产品有效保鲜的行业瓶颈，选择同种属PPO结构相近但其特异性底物不同的莲子、莲藕为研究对象，通过定点突变确定PPO催化功能域的关键氨基酸，借助分子模拟技术观测PPO与酚类底物的特异性结合过程，辅以生物谱学表征手段验证结合过程中催化功能域空间构象的变化，观察酶底物中间态结构信息，确证PPO精准识别、定位、结合特异性底物的位点和结构功能域。阐明了PPO底物特异选择性的分子机制，为植物性农副产品褐变抑制剂的研发提供理论支撑。并基于此筛选发现了一系列高效褐变抑制剂，有助于发现行之有效的新鲜果蔬保鲜方法，既有较强的代表性，同时还具有脱贫攻坚的时代特色，为促进农业经济增长和农民增收提供理论技术依据。