Mechanism and Engineering of an Autoinducer Hydrolase

描述（由申请人提供）：细菌已进化出多种机制 在恶劣的环境中生存这些保护机制之一是 生物膜的形成：一个结构化的、有组织的细菌群落。这些 生物膜对抗微生物剂的抗性显著更高，并且可导致 烧伤组织、医用导管和肺部的恶性感染 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 自体诱导物（AI）。最近，已经分离并克隆了一种AI水解酶， 这种蛋白质可以作为一种“无线电干扰”装置， 阻断细菌通讯并防止生物膜形成。非常 然而，人们对这种酶的作用机制知之甚少。AI水解酶 将根据金属含量、化学活性、反应 动力学和酶促机制。合成制备的底物，pH值 配置文件和同位素效应将与定点 诱变和替代金属掺入以探测反应 机制对于可能的治疗用途，AI水解酶应具有广泛的 底物特异性，包括多种天然存在的N-酰基-高丝氨酸 内酯。定向进化技术将被用来重塑 底物特异性和最大化总活性。试图超越 免疫原性的可能问题也将通过建立 包含AI部分的杂交蛋白的组合文库 水解酶和同源人酶的部分。如果成功， 用于产生这些混合体增量截断技术可以 适用于“人源化”其它治疗上有用的细菌酶。

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.