基于手性特点的D型苯丙氨酸脱氨酶的从头设计与创制

D-phenylalanine ammonia-lyase (D-PAL) is an important enzyme, which could catalyze and synthesize D-phe, an important pharmaceutical intermediate. It is hard to be obtained through molecular modification based on L-phenylalanine ammonia-lyase (L-PAL) up to now, due to the much structural difference between the D-PAL and L-PAL. New strategies are pursued since the molecular modification is only fit for the modification of limited structural difference of enzymes. Enzymatic de novo design based on the theory of selection of protein scaffold is a good strategy to solve this problem. However, it is a little difficult to achieve as a result of the difficulty of design. Based on pre-research foundation, this project creatively employs a new strategy of enzymatic chirality and the method of de novo design, to decrease the difficulty of design based on the L-PAL. The model of active center of D-PAL is obtained by the quantum calculation. It is then to install in the TIM scaffold with more advanced properties such as the stability. The modeled and matched structures are assayed through energetic minimum. A series of designed D-PAL gene sequences are obtained, transferred to the E. coli, expressed for verification, and further verified by the molecular simulation through the data of D-PAL activity. The aim of this project is to provide scientific basis and methodical reference for the strategy of certain chiral enzymatic design that need much structural modification.

D型苯丙氨酸脱氨酶（D-PAL）因可催化合成重要的医药中间体-D型苯丙氨酸而受到重视。目前通过分子改造相应L型酶（L-PAL）得到D-PAL的效果很不理想，关键在于D-PAL与L-PAL立体结构差异较大。分子改造一般只适合结构差异不大的酶改造，因而迫切需要开拓新的方法解决此类问题。近年来基于蛋白骨架选择理论的酶完全从头设计方法已成功应用，但因其设计难度相对较大而不易实现。本项目在预研基础上，创造性的采用酶手性特点结合从头设计方法的新策略，参考已有L型设计D型PAL，降低设计难度因而较易成功。经量子化学计算获得可靠性较高的D-PAL活性中心，装配到稳定性较好的TIM蛋白骨架中，能量优化和结构评估获得一系列D-PAL基因，利用E. coli pET高效表达D-PAL进行实验验证，比较实验数据与设计方案进行分子模拟验证。本项目旨在为部分结构差异较大的手性酶设计策略的制定提供科学依据和方法学参考。

D型苯丙氨酸脱氨酶（D-PAL）是可合成重要医药中间体D型苯丙氨酸（D-phe）的生物催化剂。本项目依据MIO催化机制及酶从头设计理论，试图设计非天然存在的D-PAL，主要结果如下：.1.以Streptomyces maritimus来源的PAM（SmPAM）为对象进行异源表达研究。在E. coli BL21中成功表达SmPAM；分析MIO催化机制并解析L-PAL活性中心结构，手性对称提取出D-PAL关键催化基团：MIO亲电基团、Tyr、Arg和Gln；量子化学计算D-PAL theozyme的过渡态获得催化构型，赋予活性中心一定自由度，通过RosettaDesign生成D-PAL theozyme催化中心三维模型集并装配至TIM桶蛋白骨架中；最后基于打分评估结果，筛选出若干D-PAL氨基酸序列。.2. 上述D-PAL在E. coli BL21中仅有8个D-PAL成功表达，其中有3个D-PAL有一定的催化作用，其最适反应pH和温度分别为6和50 °C，稳定性较好，但酶活均很低约0.1-0.2 U/mg。进一步发现D-PAL实际进攻催化底物苯环的是Asp834，但Asp834羰基O邻位活化β质子能力较MIO弱，难以获得大量的D-phe。.3.分析Anabaena variabilis来源的L-PAL（AvPAL）活性中心之外的某些氨基酸对底物催化也具有结构约束作用，故对AvPAL进行一系列突变设计，从另一途径设计D-PAL。结果显示，突变体AvPAL-Q311E/E448T间接催化D-phe的含量提高至90%以上；突变体AvPAL-N347A直接催化D-phe达到5.7 U/mg，成功设计了D-PAL。进一步构建AvPAL-N347A、PmLAAD和NH3BH3一步法偶连反应生产D-phe，在最佳反应条件下D-phe纯度达99%以上。.4.获得两个pH稳定性较好的AvPAL突变体S73N/E95K和E75A，酶活都提高了35%；同时探索了Taxus chinensis来源的MIO依赖性酶PAM（TcPAM）α-型向β-型选择性转变的规律，结果表明突变体TcPAM-N458F/L108E的β选择性较野生型酶提高了5.2倍，且催化间位带有电子供体的反式肉桂酸（t-CA）衍生物时，其β-型产物都超过90%。