乳酸菌谷氨酸脱羧酶的催化机理及其结构与功能关系研究

Glutamate decarboxylase (GAD), which catalyzes a-decarboxylation of L-glutamate or its salts into γ-aminobutyric acid(GABA), is the key rate-limiting enzyme which performs the acid resistance in many microorginisms especially lactic acid bacteria. In this study,using the GAD in the Lactobacillus brevis CGMCC 1306 as the model protein of amino acid decarboxylase, a semi-rational directed evolution method was applied to the GAD engineering to improve the enzyme activity, pH stability and thermostability, guided by the information of the spectroscopic data (X-ray diffraction and nuclear magnetic resonance), crystal structure of homologous protein or molecular dynamics simulation. The amino acid residues involved in active centre, substrate tunnel, substrate-binding pockets, the particular sites or regions relative to the thermostability or pH stability of GAD will be identified. The strategies to introduce amino acid substitutions include site-directed mutagenesis, site-saturation mutagenesis and the combination of positive mutations. On the basis of the above data, kinetic characterization, molecular simulation and molecular docking are applied to analyze the structure information of the mutant, the catalytic reaction mechanism, the conformation of enzyme catalytic active centre and the key amino acid residues which participate in the reaction. The interaction among the enzyme structure, conformation, flexibility, catalytic activity and activity regulation is also investigated. The research not only plays an important role in elucidating the catalytic mechanism of amino acid decarboxylase and the relationship of structure and function, but also provides the theory foundation and methodology instruction about understanding acid resistance mechanism of microorginasim and GABA fermentation regulation.

谷氨酸脱羧酶(GAD)能催化谷氨酸α-脱羧生成γ-氨基丁酸，是许多微生物尤其是乳酸菌抗酸系统中的关键酶。研究拟以乳酸菌中GAD作为氨基酸脱羧酶的模型蛋白，采用"半理性设计"思路，以波谱学（X射线衍射法、核磁共振等）数据、同源蛋白的晶体结构以及分子模拟信息为指导，针对位于或邻近酶分子的活性中心、底物结合口袋、底物通道附近以及与稳定性相关的特定位点/区域，采用定向进化的蛋白质工程手段进行分子改造，提高酶活力、稳定性等性能。根据酶催化反应动力学数据，结合分子对接、分子动力学模拟以及谱学数据分析突变酶的结构信息、酶活性中心的构象以及参与反应的关键残基等信息，探讨GAD的催化反应机制，阐明酶结构－构象－柔性－催化活力－活性调控间的关系。此研究不仅对阐明氨基酸脱羧酶的催化机理以及揭示其结构与功能关系具有重要意义，同时也为微生物抗酸机制的研究以及γ-氨基丁酸的发酵调控提供理论依据和方法学指导。

谷氨酸脱羧酶（GAD；EC 4.1.1.15）能专一性的催化L-谷氨酸的α-羧基脱羧生成γ-氨基丁酸（GABA）。GABA是哺乳动物中枢神经系统中的一种重要的抑制性神经递质，具有重要的生理功能。以Lactobacillus brevis CGMCC1306 GAD基因的pET28a(+)-GAD1407 重组质粒为模板，进行error-prone PCR扩增, 通过高通量比色法筛选得到催化活力显著提高的突变酶Q51H。然后在Q51位点进行饱和突变，确定了该位点的最佳氨基酸替换类型为Q51H。突变酶Q51H酶学参数测定表明，该突变对酶的底物亲和力（KM）影响较小，转化数（kcat）是野生酶的2.63倍，催化效率（kcat/KM）是野生酶的2.56倍。由于拟南芥GAD催化最适pH在5.5-7.0之间，通过比对拟南芥GAD与GAD1407的三维结构，选择S307进行定点饱和突变，突变酶S307N在pH 大于5.0时，催化活性显著高于野生酶。构建突变N-末端缺失的 GAD，并证明N-末端区域对GAD1407的正确折叠和可溶表达至关重要。通过MODELLER构建了GAD1407的三维结构模型，对酶活性中心分析后发现Phe65和Thr215构成了疏水的底物入口，Lys279、Asp248、His278、Ser127以及PLP磷酸基团附近的α-螺旋对辅酶PLP在活性中心的定位和正确取向有重要作用。以MODELLER构建的GAD模型为蛋白质受体，在GAD催化机制的指导下构建了模拟反应过渡态的偕二胺中间体模型，然后采用ROSETTALIGAND程序将该底物模型对接进入酶的活性中心。通过对接分析，发现Gln166和Thr64与底物分子形成氢键作用，是维持底物在酶活性中心正确位置和取向的关键。L-谷氨酸的α-羧基基本处于与PLP吡啶环垂直的方向，PLP的吡啶环以磷酸基团为轴翻转了约6º，再现了脱羧反应的特点。