米曲霉果糖基转移酶溶液结构、催化机制及分子改造研究

Enzymatic three-dimensional structure in solution and catalytic mechanism is the frontier scientific issues in the fields of biological catalysis. Solution structure of the fructosyltransferase from Aspergillus oryzae have not been resolved, so substrate recognition and transglycosylation reaction mechanism of the enzyme in solution are not clear. In this study, the synthesis of sucrose-6-acetate from sucrose and glucose-6-acetate catalyzed by the fructosyltransferase AoFT will be as a model reaction, and detailed studys on the solution structure and the mechanism of substrate recognition will be carried out by liquid-state heteronuclear and multidimensional NMR chromatography to clarify domain functions, active sites and mechanisms of substrates recognition of AoFT, combined with biochemical mutation, circular dichroism, fluorescence spectroscopy and different scanning calorimetry. Through the analysis of the backbone dynamics of AoFT, the relations between dynamics changes of its conformation and reaction kinectics in solution will be established, and catalytic mechanisms of AoFT in solution will be elucidated. Molecular design will be performed based on the 3D structure of AoFT in order to broaden the substrate selectivity and improve the catalytic efficiency of the enzyme. The research results will clarify the catalytic mechanism of fructosyltransferases, and have important theoretical significance and practice value for directed designs of the glycosyltransferase and biosynthesis of glycosyl-based production of chemicals.

酶在溶液中三维结构及其催化机制是当今酶催化研究前沿科学问题。米曲霉果糖基转移酶溶液结构至今尚未得到解析，因此溶液中该酶与反应底物识别及转糖基反应机制并不清楚。本课题以米曲霉果糖基转移酶AoFT催化蔗糖与葡萄糖-6-乙酸酯生成蔗糖-6-乙酸酯为模式反应，采用异核多维液相核磁共振技术解析AoFT溶液结构及其与底物的相互作用，结合生化突变、圆二色谱、荧光色谱和差示扫描量热方法，分析其结构域功能，确定AoFT与底物结合的活性位点精细结构，揭示溶液中AoFT催化底物的识别机制；通过研究AoFT主链动力学性质，深入分析其溶液构象的动态变化与动力学之间关系，阐明溶液中AoFT催化作用机制；在获得AoFT三维结构基础上进行分子设计，拓宽AoFT对底物选择性，进而提高酶催化效率。研究成果将揭示果糖基转移酶溶液结构及催化机制，在糖基转移酶定向改造以及糖基化学品生物合成等方面具有重要理论意义和使用价值。

米曲霉果糖基转移酶溶液结构至今尚未得到解析，因此溶液中该酶与反应底物识别及转糖基反应机制并不清楚。本课题利用核磁共振技术初步解析了果糖基转移酶AoFT N末端催化结构域（AoFT∆N）的溶液结构，确定了AoFT∆N属于五叶推进器结构，活性中心（Asp164、Asp39和Glu164），保守残基（His80, Pro142和Ile143）和底物结合残基（Gln57、Ser99 和Tyr282）都位于AoFT∆N分子内部凹槽周围，表明该凹槽是底物结合和发生催化反应的区域。详细研究了双标记AoFT∆N的酶催化性质，双标记蛋白具有较好酶催化活性。在果糖基转移酶AoFT∆N溶液结构基础上，初步研究了AoFT∆N的主链动力学性质。AoFT∆N不同状态下的动态特征有一定差异，其中底物类似物结合态和未结合底物AoFT∆N表现出明显的构象交换，位于催化活性中心（Asp164、Asp39和Glu164）及邻近区域的残基（Gln57、Ser99 和Tyr282）多具有微秒到毫秒时间尺度的构象交换，部分残基还存在皮秒到纳秒时间尺度的内部运动。以催化蔗糖与葡萄糖-6-乙酸酯生成蔗糖-6-乙酸酯为模式反应，推测了果糖基转移酶AoFT保留型糖苷酶反应机制，并通过酶催化分子动力学证实推测结论的正确性。在获得 AoFT∆N三维结构基础上进行分子设计，三个突变体N38L、S99A和Y282A具有较低Km和较高Vmax，相对于野生型酶具有较高酶催化活性。