Mechanism and Engineering of an Autoinducer Hydrolase

描述(申请人提供)：细菌进化出多种机制 在恶劣的环境中生存。这些保护机制之一是 生物膜的形成：有结构、有组织的细菌群落。这些 生物被膜对抗菌素的抵抗力明显更强，并可能导致 烧伤组织、医用导管和肺内顽固性感染 囊性纤维化患者。生物膜形成的一个重要触发因素是 N-酰基高丝氨酸内酯的分泌和检测，也称为 自动诱导器(AI)。最近，一个人工智能水解酶被分离和克隆。 一株芽孢杆菌。这种蛋白质可以充当一种“无线电干扰”装置， 阻断细菌交流，防止生物被膜形成。非常 然而，人们对这种酶的作用机制知之甚少。AI水解酶 将通过金属含量、化学活性、反应来表征 动力学和酶机理。人工合成底物，pH值 配置文件和同位素效应将与现场定向相结合使用 诱变和替代金属掺入来探测反应 机制。对于可能的治疗用途，AI水解酶应该具有广泛的 底物专一性，包括各种天然存在的N-酰基高丝氨酸 内酯类。定向进化技术将用于重塑 底物特异性，最大限度地提高总活性。一次超越的尝试 可能出现的免疫原性问题也将通过创建 含有AI部分片段的杂交蛋白质组合文库 水解酶和人类同源酶的一部分。如果成功，则 用于创建这些杂交的增量截断技术可以是 适用于“人性化”其他治疗有用的细菌酶。

DESCRIPTION (provided by applicant): Bacteria have evolved numerous mechanisms to survive in hostile environments. One of these protective mechanisms is the formation of a biofilm: a structured, organized community of bacteria. These biofilms are significantly more resistant to antimicrobials and can result in recalcitrant infections of burn tissue, medical catheters and in the lungs of cystic fibrosis patients. An important trigger for biofilm formation is the secretion and detection of N-acyl-homoserine lactones, also called autoinducers (AIs). Recently an AI hydrolase has been isolated and cloned from a Bacillus sp. Such a protein could act as a type of "radio-jamming" device, blocking bacterial communication and preventing biofilm formation. Very little, however, is known about the mechanism of this enzyme. AI hydrolase will be characterized in terms of metal content, chemical activity, reaction kinetics, and enzymatic mechanism. Synthetically prepared substrates, pH rate profiles, and isotope effects will be used in combination with site-directed mutagenesis and alternative metal incorporation to probe the reaction mechanism. For possible therapeutic use, AI hydrolase should have wide substrate specificity including a variety of naturally occurring N-acyl-homoserine lactones. Directed evolution techniques will be used to remold substrate specificity and maximize total activity. An attempt to surmount possible problems with immungenicity will also be pursued by creating combinatorial libraries of hybrid proteins that contain portions of AI hydrolase and portions of a homologous human enzyme. If successful, the incremental truncation technique used to create these hybrids may be applicable for "humanizing" other therapeutically useful bacterial enzymes.