Biochemistry of Phenazine Production in Pseudomonas aeruginosa

性状（由申请方提供）：吩嗪类是由铜绿假单胞菌产生的有色次级代谢产物。长期以来，吩嗪被认为是无害的，现在已知对宿主生物体具有显著和有害的生物学效应。吩嗪增强了铜绿假单胞菌的竞争能力，并帮助细菌以某种方式击败宿主防御，从而导致这种生物体引起的臭名昭著的持续感染。本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，最有可能与传统抗生素治疗相结合。本研究的总体目标是通过使用结构和机制研究来确定吩嗪抗生素生物合成的化学机制，这将填补我们理解中的空白，并允许评估开发吩嗪生物合成的特异性抑制剂的可行性。最初的三环吩嗪骨架是由七种核心酶从氯酸合成的，其中三种（PhzD，-F和-G），我们最近在晶体学和机理研究中进行了研究。这个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个关键步骤，和两个关键的吩嗪修饰酶，PhzM和PhzS，产生绿脓菌素，具有显着的生物活性的吩嗪。使用X射线晶体学，我们将确定这些蛋白质的三维结构。酶学研究将集中在每种酶的底物特异性和机械性质，有助于他们的能力，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，这些研究将解决吩嗪生物合成抑制剂的设计以及将其用作治疗剂的可行性。机会致病菌铜绿假单胞菌是一个严重的威胁，个人的免疫力受到损害的囊性纤维化，烧伤，癌症，艾滋病，和其他条件。铜绿假单胞菌占美国医院感染的10%，对于那些具有最严重潜在健康问题的人，死亡率从20%到高达70%不等。显然需要开发补充现有抗菌治疗的新策略，以更有效地对抗铜绿假单胞菌感染。

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.