Mechanism and Engineering of an Autoinducer Hydrolase

点击翻译按钮获取中文摘要

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.