突环区显著提高内切多聚半乳糖醛酸酶催化效率机理的研究

Loop structure is a research hotspot for analyzing enzymatic properties and functions of glycoside hydrolases. Endo-polygalacturonase (PG) is one of the most important enzymes in the fruit and vegetable juice industry. We obtained an endo-PG (PG8fn), which is distinguished from other reported enzymes by the highest specific activity (28122 U/mg ) known so far, and the catalytic efficiency (kcat/Km:190000ml/mg/s) of PG8fn is 6529 times higher than that of its control enzyme PG63 in this study. PG8fn shares 58% amino acid sequence identity with PG63 and the modeled structures of PG8fn and PG63 are highly similar, except that three loop regions around the catalytic channel of two enzymes show different conformations. The result suggests that the conformational characteristics of PG8fn loop regions might be the key cause of its prominent enzymatic properties. Thus the loop structure was identified as the target to explore the molecular mechanism of PG8fn’s high catalytic efficiency. Analysis of molecular docking and molecular dynamics simulation showed that loop 1，loop 2 and loop 3 are involved in the formation of active and non-active conformation. Further biological computation and mutation studies will be focused on loop-derived effects on the catalytic channel formation, steric hindrance of substrate binding and product release, and bond formation and breaking of substrate-enzyme complex. Thus the influence of dynamic conformational changes of loop regions on the catalytic reaction process will be revealed, and how specific conformation of PG8fn loop regions to form high catalytic efficiency will be determined. As a result, this study will provide theoretical and experimental basis for the improvement and optimization of industrial PG candidates in juice production.

突环区结构是解析糖苷水解酶性质功能的研究热点。内切多聚半乳糖醛酸酶(PG)是极为重要的果蔬汁工业酶种。我们发现一个催化效率目前最高的PG(PG8fn,比活力:28122 U/mg; kcat/Km:190000ml/mg/s)，它与我们分离到的另一PG(PG63)三级结构高度相似，仅在催化通道上的3个loop区呈现出不同的构象。由于二者催化效率差异显著(6529倍)，说明loop区是造成PG8fn高效催化效率的重要靶点。分子对接和动力学模拟显示3个loop区直接参与形成酶分子活性及非活性构象。以此为基础开展生物计算和突变设计，研究loop区对酶分子催化通道形成、底物结合及产物释放空间位阻、底物-酶复合物成键断键的影响，逐级揭示loop区构象动态变化对催化反应过程的影响及作用，从而阐明PG8fn特殊loop构象形成其高催化效率的机理，为果汁工业PG的性质改良提供切实可行的理论基础和实验依据。

本研究通过系统研究活性T3 loop区上底物结合位点、二级结合位点和非活性位点对酶催化效率的影响，揭示活性loop区在酶催化过程中的作用，并以PG8fn T3 loop区的结构特征指导同类酶分子改良。PG8fn与已报道晶体结构的多聚半乳糖醛酸酶CluPG1 (PDB:2IQ7)催化口袋里的氨基酸高度相似。将CluPG1上Thr121突变成Lys后，突变体T121K与底物亲和力增大，比活提高到了22,300 U/mg，几乎达到了PG8fn的水平。对该位点进行饱和突变研究，各突变体的Km值和kcat值差异显著，其中，突变体K121F和K121W的Km值较野生酶PG8fn降低，催化效率较野生酶PG8fn分别提高了44%和105%。分子动力学模拟结果显示，位于T3 loop区上的第121位残基不仅影响了酶与底物的结合模式，还直接与底物的4位GalpA相互作用，属于多聚半乳糖醛酸酶的底物二级结合位点。进一步根据PG8fn中独特氨基酸组份分析以及静态结构的通道识别，确定了位于T3 loop区上靠近通道T1的残基Thr113作为定点突变位点。突变试验结果显示各个酶催化效率从大到小的顺序与113位点的氨基酸侧链性质相关，即带正电荷氨基酸>极性氨基酸>非极性氨基酸>带负电荷氨基酸。通道分析显示突变体T113R较野生酶PG8fn通道T2和T3的占据率和优先权更高。分子动力学模拟结果表明突变体T113R T3 loop区由于盐键的断裂而导致柔性增加，继而影响了酶与底物的结合，证实了活性T3 loop区上的非活性位点可通过改变loop区柔性变化而影响酶催化效率的分子机制。基于PG8fn T3 loop区上成对的阳离子-π相互作用力这一结构特征，通过对PG63序列和结构分析结合分子动力学模拟轨迹的阳离子-π相互作用力占有率计算结果，确定8个可能在PG63中形成阳离子-π相互作用力的位点。其中，突变体H58Y较野生酶PG63的催化效率和Tm值分别提高144倍和3.9 °C。分子动力学模拟结果表明位于突变体H58Y T3 loop区上成对的阳离子-π相互作用力(Arg89-Trp90/Arg89-Tyr58)增加了邻近的T3 loop2上保守残基His158的刚性，从而很好的指引长链寡糖不断的向催化口袋里传送，增加反应速率和转换数，从而提高催化效率的分子机理。