具有新颖催化活性的细胞色素P450酶TxtE和甲基转移酶TleD的晶体结构和功能研究

The target proteins are two enzymes having novel catalytic activity which were found recently in the biosynthesis pathway. TxtE, which is a cytochrome P450, catalyzes direct nitration reaction and TleD, which is an S-adenosylmethionine-dependent methyltransferase, can catalyze terpene cyclization reaction. Unlike monooxygenase reaction, TxtE is the first reported enzyme that catalyzes a direct nitration of L-tryptophan and its analogues specifically in a biosynthetic pathway and thus it can potentially be developed for industrial applications. However, the reaction mechanism, the substrate key structural features and the reaction regioselectivity of TxtE are not yet fully understood. TleD is the first reported methyltransferase that can catalyzes terpene cyclization reaction, and shows no homolog with known monoterpene cyclase. Results from LC-MS and NMR show TleD catalyzed terpene cyclization mechanism is different from the known terpene cyclase. TxtE and TleD owned typical sequence features of cytochrome P450 and methyltransferase, respectively. And they also do not contain other unknown function domains, so it is very surprising for the new found function features. This project intends to resolve crystal structure of these two enzymes, as well as their substrate complex combined with series of experiments to clarify reaction mechanism of TxtE and TleD.

本项目的研究对象是两个近期被鉴定的拥有新颖的生物催化功能的酶：催化直接硝化反应的细胞色素P450酶TxtE和具有单萜环化酶活性的S-腺苷甲硫氨酸依赖的甲基转移酶TleD。不同于通常的单氧化酶反应，TxtE是在生物合成途径中第一个被报道的催化直接硝化反应的生物酶，可直接硝化L-色氨酸及其类似物，有潜在的工业应用价值。但TxtE的反应机制，底物的关键结构特征和反应的区域选择性等问题都尚未完全了解。TleD是第一个被发现的同时具有单萜环化酶活性的甲基转移酶，与已知的单萜环化酶没有同源性，且催化萜环化的机制不同于已知的萜环化酶。TxtE和TleD分别具有典型的细胞色素P450和甲基转移酶的序列特征，也不包含未知功能结构域，因此它们所拥有的新功能十分令人意外，且不能通过已有的知识来解释。本项目拟通过解析上述两种酶及它们的底物复合物并结合一系列功能实验，阐明TxtE和TleD的反应机制等问题。

本项目的研究目标是以催化直接硝化反应的细胞色素P450酶TxtE和具有单萜环化酶活性的S-腺苷甲硫氨酸依赖的甲基转移酶TleD为研究对象，运用结构生物学、生物化学等手段，开展蛋白质的结构与功能研究，阐明TxtE和TleD的反应机制等问题。在本项目的资助下，课题组还开展了噬菌体NrS-1 DNA聚合酶，霍乱弧菌群体感应关键调控蛋白VqmA的结构和功能研究。本课题组成功解析了TxtE和TleD的晶体结构，并通过定点突变，分子模拟，分子对接等手段研究了晶体结构和功能的相互关系。噬菌体NrS-1 DNA聚合酶具有聚合酶和引发酶双功能活性。通过解析该聚合酶的核心结构域，并分析了进化关系、反应机制、酶催化特性，在底物结合、底物特异性以及参与催化的关键残基，为噬菌体家族聚合酶/引发酶双功能酶提供了结构模型。霍乱弧菌群体感应关键调控蛋白VqmA的研究为基于相关蛋白开发新型抗生素提供了结构基础。