氨基酸与Brevibacillus laterosporus抗菌肽的构效关系及其调控酶的催化机理研究

Antimicrobial peptides secreted by Brevibacillus laterosporus (BL AMPs) is currently the most potential new bio-preservative, and its structure is the critical factor for the biological function. Previous studies have shown that the changes of amino acids (AA) from BL AMPs could result in varied antibacterial properties. However, the effect of AA on the function of BL AMPs has not been clearly elucidated, especially its molecular mechanism. Therefore, the present project intends to elucidate the interaction among AA, the structure of BL AMPs and the antimicrobial properties, to explore the function and catalytic mechanism of adenylation domain which is a key enzyme for AA regulation. Some major contents are included in our research proposal: first, determining the structural changes of BL AMPs affected by AA, then analyzing the effect of structural changes on antimicrobial properties, in order to clearly elucidate the interaction between AA and BL AMPs. Second, the adenylation domain will be obtained by recombinant technique, and then the enzymatic characteristics and specificity recognition ability of the recombinant enzyme towards AA substrate will be analyzed. Third, the three-dimensional structure, as well as the catalytic active sites will be predicted, in order to elucidate the catalytic mechanism. Some mutants having key catalytic site will be constructed under gene mutation, and the key site mutation structure will be analyzed by bioinformatic tools. The role of key site of adenylation domain will be well explained by constructing the "gene-enzyme-structure" research system. Finally, the molecular mechanism of the specificity recognition of AA substrate catalyzed by adenylation domain from Brevibacillus laterosporus will be revealed. The implementation of the project can lay a theoretical foundation for the in-depth study of the BL AMPs biosynthesis and its rational transformation.

侧孢短芽孢杆菌抗菌肽（BL AMPs）是目前最具应用潜力的新型生物防腐剂，抗菌肽结构能决定其生物功能，具有重要研究意义。前期研究发现BL AMPs的氨基酸（AA）组成变化能严重影响其抑菌性能，但其如何影响以及产生此影响的生物机制尚未明确。本课题将系统探究AA与BL AMPs结构及其抑菌性能间的构效关系，挖掘该菌调控抗菌肽AA组成关键酶——腺苷酰化酶的功能及其调控机制。解析抗菌肽的结构变化，并剖析结构变化对抑菌性能的影响规律，阐明AA组成与BL AMPs功能间的构效关系；采用重组技术获取腺苷酰化酶，解析酶学特性，重点评价其特异性识别AA的能力；解析重组酶的晶体结构并获取关键活性位点，预测酶对AA的识别机制；构建核心位点突变体，建立“基因-酶-结构”研究体系解读核心位点的作用。最终，揭示腺苷酰化酶特异性识别AA的分子机制。项目实施可为深入研究BL AMPs生物合成机制及其理性改造奠定理论基础。

抗菌肽是控制微生物污染、保障食品安全的新型生物防腐剂。Brevibacillus laterosporus抗菌肽（BL AMPs）是目前最具应用潜力的新资源，其生物学功能主要由其结构决定。本项目组前期选育出一株优产BL AMPs（命名为Brevilaterin）的菌株S62-9，并发现氨基酸能严重影响Brevilaterin的抗菌性能，但如何影响以及产生此影响的生物机制尚未明确。因此，本项目首先考察了外源氨基酸对Brevilaterin抗菌活性和组分构成的影响，发现多种氨基酸能提高其抗菌活性、改变组分构成甚至还能促进产生新结构的Brevilaterin。利用制备液相纯化出5个新结构的Brevilaterins（Brevilaterin V1、V2、V4、V5和V6）。通过MALDI-TOF/TOF-MS、2D-NMR及圆二色谱等解析其结构，发现其氨基酸数量不变，但有1~3个氨基酸残基不同于原组分。它们具备广谱、高效的抗菌活性、优良的环境适应性，且Brevilaterin V1和Brevilaterin V2还有非常优异的溶血活性。其次，从S62-9全基因组中挖掘出Brevilaterin合成中决定特异性识别氨基酸底物的关键酶——腺苷酰化酶基因，采用分子生物学手段克隆出14个腺苷酰化酶基因并实现异源表达。通过antiSMASH预测出重组酶的识别底物，再利用钼锑抗分光光度法及焦磷酸盐检测试剂盒确定其实际识别底物，发现Bre270A实际识别底物与预测底物完全一致，Bre2652A实际识别底物与预测底物完全不同，而其余酶的实际识别底物范围均宽于预测底物。最后，以Bre2691A为例，利用SWISS-MODEL构建重组酶的同源模型。解析三维结构，发现其主体结构由Acore和Asub两个亚基组成，它们通过氢键来捕获底物，且亚基之间还含有一个由10个氨基酸残基构成的“特异性代码”，它对该酶识别底物起到关键作用。再利用AutoDock vina软件对Bre2691A和底物进行分子对接，挖掘其“特异性代码”上的3个关键残基，采用定点突变技术单突变和三突变上述位点，验证其对该酶特异性识别氨基酸底物的重要作用，初步阐明腺苷酰化酶的底物识别机制。项目研究为将来Brevilaterin生物合成的研究及人工合理调控Brevilaterin的生产奠定了理论基础。