新家族糖酯酶H22的催化机制及适冷机制研究

Carbohydrate esterases play an important role in the degradation and cycle of marine organic carbon. H22 is a novel cold-adapted carbohydrate esterase isolated from the surface seawater from the King’s Fjord, Svalbard. Based on the phylogenetic analysis, H22 and its homologs may represent a new family of carbohydrate esterases. Homologous sequences to H22 are present in many genomes of marine bacteria, but these predicted sequences are not characterized up to date. Biochemical analysis showed that H22 has degradation activity on various acetylated oligosaccharide substrates, and with the highest activity towards 1,2,3,5-tetra-O-acetyl-D-xylofuranose. Due to the lack of structural information, the mechanism for substrate recognition and catalysis of H22 is still unknown, and the mechanism for the cold adaption of H22 to arctic low-temperature environment is also unknown. To answer this question, we will overexpress, purify, and biochemically characterize this protein in this project. Then, we will crystallize H22 and H22 bound to substrate. Based on structural analysis, mutational analysis combined with biochemical analysis, the mechanism for catalysis of H22 will be elucidated. Moreover, based on structural analysis of H22 and reported mesophilic or thermophilic carbohydrate esterases and mutational analysis, the key residues or key structural element related to the cold adaption of H22 will be determined. Thus, the mechanism for the cold adaption of H22 to arctic low-temperature environment will also be elucidated.

糖酯酶在海洋有机碳的降解和循环中发挥重要作用。H22是一个分离自北极表层海水的新型低温糖酯酶。系统发育显示，H22及其同源序列可能代表了糖酯酶的一个新家族。H22的同源序列存在于多个海洋细菌的基因组中，但是这些序列均未被研究。生化性质分析表明，H22对多种乙酰化寡糖具有降解活性，其中对乙酰化木糖的降解能力最强。关于H22识别并催化底物的分子机制还不清楚，其适应极地低温环境的机制也不清楚。为了阐明H22的催化机制和适冷机制，本项目首先对H22进行异源表达和分离纯化以及生化性质分析，揭示出H22的催化特性。随后，对H22及其与底物复合物的晶体结构进行解析和分析，结合突变和生化分析，揭示出H22降解乙酰化寡糖底物的分子机制。同时，对低温酶H22和中温或高温糖酯酶的结构进行比较分析并结合突变分析，确定影响H22适冷性的关键结构元件或氨基酸残基，揭示出糖酯酶H22适应极地海洋低温环境的结构基础。

从极地海洋细菌Arcticibacterium luteifluviistationis SM1504T基因组中筛选到了一个新型乙酰木聚糖酯酶，AlAXEase。AlAXEase能够水解乙酰化木寡糖和木聚糖。系统发育分析表明，AlAXEase及其同源序列可能代表了一个新的SGNH型糖酯酶家族。AlAXEase的最适酶活温度为30℃，且在0℃保留超过70%的活力，同时AlAXEase具有较高的热稳定性（Tm为56℃）。我们解析了AlAXEase的晶体结构以研究其适冷机制。AlAXEase在单体水平上与其中温同源蛋白具有相似的非共价相互作用，并且在溶液中以四聚体形式存在，这可能是其具有高热稳定性的原因。然而，由于减少的疏水相互作用以及较多不稳定残基（天冬酰胺和赖氨酸）的存在，AlAXEase中含有催化残基Asp200和His203的长loop具有很高的柔性，进而导致活性位点区域柔性较高。结构和酶动力学分析结合不同温度下分子动力学模拟表明催化loop通过调节催化残基Ser32和His203之间的距离，促进了其适冷性。本研究揭示了热稳定糖酯酶AlAXEase采用的一种新的适冷策略，为乙酰木聚糖酯酶适冷机制的研究提供了基础。本项目还资助了海洋新型单酰甘油酯脂肪酶的催化特性研究、热液区产蛋白酶高温菌株胞外蛋白酶多样性分析、海洋产木聚糖酶细菌的多项分类研究及酶生化性质分析、海洋细菌产生的新型褐藻胶裂解酶特性研究等项研究。项目已资助发表SCI论文5篇。