小白链霉菌聚二氨基丙酸合成关键酶的结构域功能与催化机制研究

Homopoly(amino acid)s have broad application prospect. The scarcity of homopoly(amino acid)s synthase and the analysis of its catalytic mechanism are the bottleneck to discover new homopoly(amino acid)s. In the early stage of this project, a new type of homopoly(amino acid)s, poly(L-diaminopropionic acid) (PDAP), was identified. It is a potential natural food preservative since its antimicrobial spectrum complements ε-PL. The key enzyme in the biosynthesis of PDAP, PDAP synthetase (PDAPs), is a novel single-module nonribosomal peptide synthetase (NRPS), but little is known about how PDAPs catalyzes the polymerization of amino acid monomers. This project set the key enzyme PDAPs from Streptomyces albulus PD-1 as research object, based on previous structure prediction results, we plan to obtain pure PDAPs for the first time using yeast expression, detergent solubilization and membrane protein purification techniques, verify its activity in vitro and investigate the enzyme properties; and to study the structural domain function of the enzyme and polymerization mode of substrate monomer through domain truncation, isotopic labeling and oxidation experiments in low-temperature conditions. This study will not only lay foundation for the biosynthesis and regulation of PDAP, but also enrich our understanding of homopoly(amino acid)s synthase, and provide a good theoretical basis for exploring and synthesizing more new homopoly(amino acid)s in the future.

聚氨基酸有着广阔的应用前景，聚氨基酸合成酶的稀缺与催化机制的解析是制约挖掘新型聚氨基酸的瓶颈。本项目前期发现了一种新型聚氨基酸—聚二氨基丙酸（PDAP），它与ε-PL抗菌谱有互补性，是一种潜在的优良生物防腐剂。催化其合成的关键酶—聚二氨基丙酸合成酶（PDAPs）是一种新型单模块非核糖体肽合成酶（NRPS），但我们对PDAPs如何催化氨基酸单体聚合的机制还知之甚少。本项目以小白链霉菌（Streptomyces albulus）PD-1中PDAPs为研究对象，结合前期结构域预测基础，拟通过酵母表达、去垢剂筛选和膜蛋白质纯化技术首次获得PDAPs纯酶，进行体外活性和酶学性质考察；采用结构域敲除、同位素标记和低温氧化等手段开展酶结构域功能和底物单体聚合模式研究。本研究将为PDAP的合成和调控奠定理论基础，也将丰富我们对聚氨基酸合成酶的认识，为未来发掘和人工合成更多的新型聚氨基酸提供良好的理论依据。

聚氨基酸有着广阔的应用前景，项目前期发现了一种新型聚氨基酸—聚二氨基丙酸（PDAP）。催化其合成的关键酶—聚二氨基丙酸合成酶（PDAPs）是一种新型单模块非核糖体肽合成酶（NRPS），但我们对PDAPs的催化机制还知之甚少。该项目通过构建带有高特异性RGS-His标签、高效纯化标签ZZ tag的表达单元，利用含有链霉菌组成型表达强启动子-红霉素抗性基因PermE*的pIB139质粒，获得了适合重组PDAPs表达的载体。利用大肠杆菌S17-1，建立了高效的链霉菌接合转移方法，转化结果表明接合子能够正常产孢子，进而验证了重组PDAPs的活性。利用PKS/NRPS Analysis，PolyPhobius等程序预测和分析了PDAPs的AT功能域、穿膜区域、C1、C2及C3功能域，并进一步通过优化AT功能域序列、选择合适诱导条件和透析等方法，实现了AT功能域蛋白的可溶性纯化。A结构域识别氨基酸底物并活化，而T结构域对活化的底物进行亲核进攻，生成固定的氨酰基硫酯，C1、C2及C3功能域对底物单体的聚合起着重要作用。该项目实验的设计具有原创性和实用性，为膜蛋白质的表达纯化等问题提供了新的思路，对膜蛋白功能域的选择性表达具有指导意义，同时为PDAP的合成和调控提供了一定的理论基础，丰富了我们对聚氨基酸合成酶的认识。

内容获取失败，请点击重试