Biochemistry of Phenazine Production in Pseudomonas aeruginosa

描述(申请人提供)：吩嗪是由铜绿假单胞菌产生的有色次级代谢物。长期以来，吩嗪类药物一直被认为是无害的，但现在人们知道它们对宿主生物具有显著的有害生物效应。吩嗪类药物增强了铜绿假单胞菌的竞争能力，并帮助细菌智取宿主防御，从而导致由该细菌引起的臭名昭著的持续感染。这项研究的长期目标是评估禁用吩嗪生物合成途径的可行性，并评估这是否可能是控制铜绿假单胞菌的有效策略，最有可能与传统的抗生素治疗相结合。这项研究的总体目标是通过使用结构和机理研究来确定吩嗪类抗生素生物合成的化学机制，以填补我们在理解上的空白，并允许评估开发吩嗪生物合成的特定抑制剂的可行性。最初的三环吩嗪主链是由分支酸通过七种核心酶合成的，其中三种酶(PhzD，-F和-G)是我们最近在结晶学和机械学研究中研究的。该项目的具体目标是评估另外三种酶的酶学和结构生物化学。我们将研究PhzE，它催化吩嗪生物合成的第一步，以及两个关键的吩嗪修饰酶PhzM和PhzS，它们产生具有显著生物活性的吩嗪。利用X射线结晶学，我们将确定这些蛋白质的三维结构。酶学研究将重点放在底物专一性和每种酶的机械特性上，这些特性有助于它们有效地产生吩嗪途径中间体。这些结果将为未来的研究提供一个框架，这些研究将涉及吩嗪生物合成抑制剂的设计和将其用作治疗药物的可行性。条件致病菌铜绿假单胞菌对因囊性纤维化、烧伤、癌症、艾滋病和其他疾病而免疫力受损的个人构成严重威胁。铜绿假单胞菌占美国医院感染的10%，在那些潜在健康问题最严重的人中，死亡率从20%到高达70%不等。显然，需要开发补充现有抗菌疗法的新战略，以更有效地抗击铜绿假单胞菌感染。

DESCRIPTION (provided by applicant): Phenazines are pigmented secondary metabolites produced by Pseudomonas aeruginosa. Long believed to be innocuous, phenazines are now known to have significant and deleterious biological effects on host organisms. Phenazines enhance the ability of P. aeruginosa to compete and aid the bacteria in outwitting host defenses in ways that contribute to the notoriously persistent infections caused by this organism. The long term objective of this research is to evaluate the feasibility of disabling the phenazine biosynthetic pathway and assessing whether this could be an effective strategy for controlling P. aeruginosa, most likely in combination with traditional antibiotic therapy. The overall goals of this research are to determine the chemical mechanisms of phenazine antibiotic biosynthesis through the use of structural and mechanistic studies that will fill gaps in our understanding and permit evaluation of the feasibility of developing specific inhibitors of phenazine biosynthesis. The initial tricyclic phenazine backbone is synthesized form chorismic acid by seven core enzymes, three of which (PhzD, -F, and -G) we recently examined in crystallographic and mechanistic studies. The specific aims of this project are to evaluate the enzymology and structural biochemistry of three additional enzymes. We will examine PhzE, which catalyzes the first committed step in phenazine biosynthesis, and two key phenazine modifying enzymes, PhzM and PhzS, that produce pyocyanin, a phenazine with significant bioactivity. Using X-ray crystallography, we will determine the three- dimensional structures of these proteins. Enzymology studies will focus on the substrate specificity and mechanistic properties of each enzyme that contributes to their ability to efficiently produce phenazine pathway intermediates. The results will provide a framework for future studies that will address the design of phenazine biosynthesis inhibitors and the feasibility of using them as therapeutics. The opportunistic pathogen Pseudomonas aeruginosa is a serious threat to individuals whose immunity is compromised by cystic fibrosis, burns, cancer, AIDS, and other conditions. P. aeruginosa accounts for 10 percent of nosocomial infections in the United States and mortality rates range from 20 percent to as high as 70 percent for those with the most serious underlying health problems. New strategies that complement existing antimicrobial therapies clearly need to be developed to more effectively combat P. aeruginosa infections.