GH5_5纤维素酶loop区的架构及其对酶催化效率影响机制的研究

The mobility of flexible loops in enzymes plays an important role in catalytic efficiency by affecting the enzyme configuration, substrate binding and product release, and thus represents a major bottleneck in efficient degradation of substrate into products. However, most studies on the catalytic efficiency of glycoside hydrolases so far focused on the active regions in catalytic pockets. In the present project, computer-aided design (CAD) and experimental work will be combined to study the loop framwork of GH5_5 cellulases with significant application potentials and their effects on enzyme catalytic efficiency. Bioinformatic tools will be applied to collect the evolution information of loop framwork and functions of GH5_5 cellulases, to find out the characteristic pattern of sequence or structure in loops related to catalytic functions, and to establish the sequence profile of enzyme-specific loops. Molecular dynamic simulation (MDS) of enzyme and enzyme-substrate complex will be conducted to identify the key loops and amino acid residues related to enzyme configuration alteration. Moreover, nuclear magnetic resonance (NMR) spectroscopy will be used to determine the dynamics of loops during the interaction of GH5_5 cellulases and substrate. On the basis of the results of sequence and structure alignment, computational simulation and NMR, loops will be replaced or deleted, and site-directed mutagenesis will be conducted to the key residues. The experimental work will reveal the catalytic mechanism of loops on GH 5 glycoside hydrolases and even proteins with similar (b/α)8 barrel fold structures. As results, loops of high efficiency will be identified, combined and synthesized, and will be used to improve the catalytic efficiency of GH5_5 cellulase. This study will cast an insight in protein engineering of glycoside hydrolases of GH5 for greater catalytic efficiency and be of importance in both research and application.

目前对酶催化效率的研究集中在活性口袋区，但我们前期研究发现通过不同催化效率的酶之间loop的替换，可以有效提高酶的催化效率，预示酶loop区也是参与决定着酶的催化效率的关键因素。本项目开展有重要应用价值的GH5_5家族酶(纤维素酶)loop区的架构及对酶催化效率的影响机制研究。通过生物信息学算法提取不同催化效率的GH5_5纤维素酶loop区结构和功能信息，找到基于序列或结构的特征模式。通过分子动力学模拟和核磁共振（NMR）分析，确定loop的运动状态和变化特征。以我们获得的序列一致性高但催化效率差5倍的T. emersonill Egl5A和B. antennata Cel5为核心材料，进一步通过loop替换、删除、突变及loop的全新组合设计等，开展比较研究和实验验证，最终揭示loop区影响酶催化效率的重要机制，为指导GH5家族糖苷水解酶催化性能改良提供新途径。

研究酶loop区的变化特征，对于探究loop区对酶的催化功能影响机制、指导进行酶的改良及设计新酶有着重要的意义。本项目基于蛋白不同结构层次的分析，采用半桶融合、二级结构单元替换、定点突变等技术，系统的研究了GH5纤维素酶结构原件及特定氨基酸位点与酶功能之间的关系，阐明了纤维素酶的稳定性及催化机制。纤维素酶SoCel5与TeEgl5A的序列一致性为51%，基于二者的二级结构特点，进行结构重组。杂合蛋白H8和H9在55°C下的半衰期分别达到亲本蛋白SoCel5的650和155倍。此外，H8和H9的比活值分别为720U/mg和920 U/mg，均高于两个亲本SoCel5(350 U/mg)和TeEgl5A(600U/mg)。分子动力学模拟结果表明，N端区域中Arg52点与周围氨基酸的氢键作用力以及α2和α3结构之间的疏水堆积力对嗜热酶的热稳定性发挥重要作用，使蛋白结构在高温下保持稳定。进一步，将TeEgl5A的N端半桶结构 (βα)1-4和3/8桶结构(βα)1-3分别替换至BaCel5，构建两个杂合酶BaCel5127和BaCel5167，其比活性和催化效率提高了1.0–6.7倍。结构模拟的结果表明，BaCel5127和BaCel5167催化通道整体构型较BaCel5发生明显变化，有利于底物的结合和产物的释放，同时催化残基之间的距离也分别缩小了2.5Å和1.8Å，有利于对底物的切割。此外，TeEgl5A的N端半桶结构还为杂合酶引入了更多的氢键作用力，促进酶与底物的结合。通过本研究，证明了TeEgl5A的N端半桶结构对酶的催化活性的重要影响，同时成功的对BaCel5的催化效率进行了改良。在此基础上，对GtCel5的Asn233位点进行饱和突变研究，该位点参与loop 6上的发卡结构的形成。通过对突变体性质分析发现，N233G和N233A比活力和催化效率较野生型提高了26–70%。项目以TeEgl5A和SoCel5作为对照研究，其突变体的活力结果为Gly>Ala>Asn，与GtCel5突变结果一致。同源建模、分子对接、分子动力学模拟技术分析结果表明，233位点间接地影响了催化残基Glu267周围的氢键作用力网络。此外， Ala和Gly为233位点增加了新的氢键作用力，其与底物的结合能也显著降低。该研究阐明了loop 6区对GH5纤维素酶催化活性的影响机制。