莱茵衣藻甘油三酯（TAG）合成的关键酶PDAT构效关系的研究

It is well known that microalgae is the most potential bioresource to provide lipids. However there is a big obstacle to be overcome is microalgal lipid accumulation under stress condition not concomitant with biomass increase. Phospholipid:diacylglycerol acyltransferase (PDAT) has been identified as one of the key enzymes in chloroplast to be responsible for TAG synthesis in microalgae recently as well as the regulator of the conversion from polar lipids to TAG. PDAT is CoA independent acyltransferase to catalyze phospholipid transfer acyl group to DAG then forming TAG. Base on the function so far PDAT is in the limelight as the target to break through the bottleneck above. The recognition and catalytic mechanism of the substrate for PDAT are still veiled because PDAT is of the complication of two-substrate and two-active-site. In the preliminary study the sequence comparison and the analysis of structural domain limits of PDAT from Chlamydomonas reinhardtii (CrPDAT) and corresponding homologue enzymes have been done to predict different domains. Distinctly there are two big sequence fragments compared to homologue enzymes referred to GAP 1 and GAP 2. In the proposal the GAP 1 and GAP 2 will be identified whether they affect the activity of CrPDAT functionally. By the “frozen” strategy of tuning side chain of amino acid residue to control the stage of CrPDAT and substrate in the catalysis rational designed amino acid residues have been mutated combined with predicted domains. It will be investigated that the selectivity and catalysis of CrPDAT to acyl donors and the mechanism of CrPDAT to acyl acceptor by enzymatic activity assay and 3-dimensional crystal structures. It will shed light on the catalytic molecular mechanism of CrPDAT with two-substrate and two-activite-site from the results of this proposal. Also it will enrich the information about the molecular mechanism of PDAT like enzymes. Consequently the fundamental theory of rationally design CrPDAT will be available in order to achieve microalgal lipid accumulation accompanying with biomass increase.

微藻是最具潜力的生物油脂提供者，但现有胁迫调控无法实现生长与产油同步，亟需从源头突破。PDAT是新近发现于微藻叶绿体中负责TAG合成的关键酶，也是调节油脂间平衡的关键酶之一，能够不依赖酰基CoA完成极性脂向甘油三脂的转化，有望成为微藻油脂调控技术提升的突破口。但受限于PDAT双底物双活性中心的复杂性，其底物识别与催化机制尚不清楚。因此，本项目以模式藻株莱茵衣藻PDAT蛋白为出发，以序列比对与非序列结构域预测为指导，探索其两个未知功能插入片段对催化活性的影响；针对双底物双活性中心特点，基于氨基酸支链活性的调控实现酶催化过程反应的“冻结”策略，通过酶活及蛋白/蛋白-底物复合物3D结构解析结合，探索PDAT底物识别、水解以及酰基转移活性的分子机制。本项目将首次提供双底物双活性中心的PDAT的催化机理，加强酰基转移酶的分子水平认识，同时为理性改造PDAT、实现生长产油同步提供理论基础。

微藻是最具潜力的生物油脂提供者，但现有胁迫调控无法实现生长与产油同步，亟需从源头突破。PDAT是新近发现于微藻叶绿体中负责TAG合成的关键酶，也是调节油脂间平衡的关键酶之一，能够不依赖酰基CoA完成极性脂向甘油三脂的转化，有望成为微藻油脂调控技术提升的突破口。但受限于PDAT双底物双活性中心的复杂性，其底物识别与催化机制尚不清楚。本项目研究主要围绕ScPDAT作为CrPDAT将GAP区去掉后的突变ΔTMCrPDAT的酰基供体底物识别、水解和酰基受体活化、酰基转移分子机理研究，通过克隆、表达、纯化获得ScPDAT蛋白，利用TLC半定量法以及GC对ScPDAT酶活进行表征，揭示了ScPDAT 酰基供体底物选择性机制，发现ScPDAT 倾向利用带正电荷的磷脂（PC/PE）作为酰基供体；不能识别16:0/16:0 DAG； 更倾向利用不饱和脂肪酸作为酰基受体，同时也偏好不饱和脂肪酸作为酰基供体。推测酰基链上双键的存在利于ScPDAT 的识别。此外，发现ScPDAT不具有lipsae功能，而CrPDAT有，得出与CrPDAT中的GAP1区域有关，间接获得GAP区域与酰基受体接受酰基的的功能相关。另外，发现ScPDAT在表达过程中被糖基化修饰，并确定了其糖基化位点，而糖基化修饰对ScPDAT功能没有影响。对于非酰基CoA依赖型的双底物酰基转移酶利用缺刻缘绿藻源GPAT（LiGPAT）的蛋白晶体结构，结合分子对接技术和突变体酶活实验从结构上确定了LiGPAT 对 18:1-CoA偏好性高于16:0-CoA，LiGPAT上 pocket-2 通道内部的氨基酸涉及到酰基链的识别。以上研究结果为双底物酰基转移酶底物识别机制提供了分子水平的信息。