脂肪酶SMG1底物特异性的结构基础研究

Lipases are a class of enzymes that have found broad interests for industrial applications, but substrate selectivity of native lipases cannot satisfy all requests in industries. At present, poor understanding for structure-function relationships of lipase often results in failure to improve the substrate selectivity via protein design. The lipase from Malassezia globosa (SMG1), considerably differing from most of lipases in substrate selectivity, is strictly specific for monoacylglycerol and diacylglycerol, but not triacylglycerol. SMG1 can serve as a paradigmatic example for understanding the structure-function relationships of lipases, which would provide cues for the rational design of mutants with expected changes in substrate selectivity. We have solved the crystal structures of SMG1 in the closed conformation and several regions that participate in substrate recognition are identified, but the structural basis for its unique substrate selectivity is still not very clear. In order to address the molecular mechanism of this unique feature, the crystal structure of SMG1 in the open conformation will be resolved in complex with covalent inhibitors. Strategies of computational structural biology, including homology modeling, molecular docking and molecular dynamics simulations, will be performed to target amino acids that may play important roles in substrate selectivity of SMG1. The corresponding mutants will be expressed in Pichia pastoris and purified by anion exchange chromatography to study their changes in catalytic properties. The combination of structural insights and catalytic properties data from wild-type and site-directed mutants would shed light on the mechanism underlying substrate selectivity of SMG1. These results would provide a solid foundation for understanding the structure-function relationship of SMG1 and pave the way for site-directed mutagenesis experiments to improve the performance of enzymes for their applications.

天然脂肪酶的底物特异性通常难以满足工业需要，而对其结构与功能关系认识不足制约了脂肪酶分子改造的效果。球形马拉色菌脂肪酶（SMG1）是一种独特的脂肪酶，它仅能水解甘油单酯和甘油二酯，而不水解甘油三酯，因此，SMG1是研究脂肪酶底物特异性的理想模型。在前期研究中，我们解析了SMG1闭合构象的晶体结构，发现了参与底物识别作用的多个关键区域，但尚未完全阐明SMG1底物特异性的结构基础。本项目拟：1）通过复合物结晶的方法获得SMG1开放构象的精确结构；2）利用分子对接和分子动力学的手段找出可能影响SMG1底物特异性的关键氨基酸；3）结合定点突变实验研究SMG1结构与特异性之间的内在联系，最终明确SMG1底物特异性的结构基础。本项目将深入研究SMG1的结构与功能关系，为SMG1的工业化应用奠定基础，同时也为其它脂肪酶底物特异性的分子改造提供借鉴和指导。

球形马拉色菌脂肪酶SMG1是一种具有独特底物选择性的脂肪酶，不但可作为脂肪酶结构与功能关系研究的重要模型，而且极具应用价值。目前，影响SMG1底物选择性的分子机制尚未完全阐明。本项目通过复合物结晶的方法获得SMG1开放构象的精确结构，同时利用结构计算模拟和定点突变实验相结合的方式来进一步的明确影响SMG1底物选择性的关键位点及其协同作用机制。