理性设计P450甲烷单加氧酶及其分子机理研究

Methane is the most important component of natural gas (70~95%, v/v) and the second largest contributing greenhouse gas besides carbon dioxide. The conversion of gas to liquids (GTL) for methane is of importance in views of the demands of energy and chemical source, as well as environmental protection. In contrast to the current industrial process of GTL of methane that is technologically complex and multistep process, biological conversion of methane has attracted great attention due to the potentials of high efficiency, high selectivity, and environmentally friend. We are interested in cytochromes P450 monooxygenases (CYP or P450s), a super family of iron-containing heme enzymes (> 21000 identified), some of which are thought of great potentials for the hydroxylation of methane besides methane monooxygenases (MMO) from methanotrophic bacteria and the related ammonia monooxygenase (AMO), the latters are suffered from the bacteria growth slow or purify difficult. So far, only one of engineered P450s, CYP153A6, is known to be capable of oxidizing methane. However, it is not a real monooxygenase because of the use of PhIO as terminal oxidant, in which the catalytic mechanism is different from the general P450s those induced by substrate recognition. In this proposal, to develop a real methane monooxygenase of P450, we reasoned that the key issue is “how does P450 recognize, fix, and oxidize methane”. Based on the structure and catalytic mechanism of P450BM3, a long chain fatty acid hydroxylase, as well as the properties of methane (non-polar, small, no anchoring group, we plan to make a methane cavity by combining site-directed mutagenesis and chemical activation strategy in the large substrate tunnel of BM3 to allow enzyme fixing and then oxidizing methane. Recently, we and others have demonstrated that the additional activator molecule could occupy partially space of BM3 to reform the substrate tunnel of BM3. The left space could accommodate the small alkanes, such as propane and ethane. However, the space is still larger than methane so that the current system of BM3 can’t oxidize methane. On the other hand, it is admitted that the substrate space can be modified by the introduced amino acid residue through site-directed mutagenesis. So, in this proposal, we will firstly use an activator molecule to reform the active site of BM3, and then further reduce the reformed cavity size by introducing appropriate amino acid residues near the active center, which will help us to make an appropriate cavity for methane in BM3. Base on this idea, in this project, we have identified some residues and planned to make saturation mutagenesis at these positions, such as A328 and 82A. The other factor that may affects reactivity is the binding strength of activator molecule with protein, which may affect the stability of the reformed cavity. We will also design and synthesize new activator molecule having high affinity with protein. The oxidation of methane will be examined by combination mutants and activator molecules. The crystal of BM3 bound with activator molecule will be prepared for understanding catalytic mechanism. Through this project, we aim to construct a new methodology for developing P450-based methane oxidation system. It will provide theoretical direction and technical support for the design, reformation, and new functions development of P450s as well as the related enzymes.

生物催化的甲烷“气液转化”对于能源、化工、和环保领域都有重要意义。天然甲烷氧化酶如MMO等结构复杂且不易于大量重组表达，一定程度上增加了通过分子工程改造来达到工业应用的困难。来源广泛、结构简单、表达技术成熟、能氧化惰性C-H键的P450，在人工甲烷氧化酶开发方面有巨大潜力和前景。然而唯一能氧化甲烷的CYP153A6变体，却只能以PhIO为氧化剂，并非通过底物识别引发O2活化的单加氧酶。本项目围绕“如何让P450识别、固定、氧化甲烷”的关键科学问题，基于脂肪酸羟化酶P450BM3的结构和催化机理、及甲烷性质（非极性、体积小、无锚定基团），拟采用定点突变与化学活化相结合，在利用活化剂分子占据部分固有底物通道的同时、引入合适残基调节剩余空间，创造一个适配度高的“甲烷口袋”，实现对其“固定”和氧化，建立基于P450的甲烷氧化新途径。为P450及有关酶学新功能的开发和设计改造提供理论基础和技术支撑。

生物催化的甲烷“气液转化”对于能源、化工、和环保等领域都有重要意义。本项目针对甲烷生物催化转化受制于天然酶如甲烷单加氧酶MMO等活性较低且不易于通过蛋白质工程进行改造问题，聚焦来源广泛、体外表达技术成熟、结构简单、且能高效催化惰性C-H键选择氧化的P450酶，拟通过蛋白质工程结合外源小分子化学活化策略开发人工P450甲烷氧化酶。本项目首先设计并合成了一类一段带有锚定基团、另一端带有助催化基团的双功能小分子（DFSM）。建立了DFSM协同驱动P450催化的人工过加氧酶体系。并通过结构生物学和理论计算模拟阐明了该体系的结构基础和催化过氧化氢活化机理。进而通过对P450酶固有底物通道组成氨基酸残基分析，采用组合活性位点定点突变技术，改造P450酶的底物空间。并协同DFSM分子对底物空间的调控效应，创造出与小分子烷烃、苯乙烯及其衍生物、芳香醚及其衍生物、萘等芳香化合物具有较高适配度的“底物口袋”。从而开发出首个过氧化氢依赖型人工P450丙烷羟化酶，并检验其对乙烷和甲烷羟化活性；首次实现P450催化的高R-对映选择性苯乙烯环氧化（ee > 98%）；开发出P450催化木质素单体脱甲基化反应新体系；进一步通过机理指导的蛋白质工程将P450过加氧酶改造为P450过氧化物酶，强化了P450酶的催化多功能性。本项目在国际上首次建立利用外源引进的双功能小分子协同P450酶催化策略，将无法直接利用过氧化氢的P450单加氧酶改造为高效过氧化酶模式，从而避免了P450单加氧酶体外催化过程中对还原辅酶NADPH和电子传递伴侣蛋白的依赖、极大简化了P450酶的催化循环、为开发基于P450酶的新型生物催化剂开辟了新的途径，为开发基于P450的甲烷氧化酶提供了新思路，为P450及有关酶学新功能的开发和设计改造提供理论基础和技术支撑。