Structural studies of bacterial quorum sensing regulator

描述（由申请人提供）：持续性细菌感染是一种 囊性纤维化患者死亡的主要原因和免疫受损 个体包括假单胞菌在内的一些革兰氏阴性菌 铜绿假单胞菌是囊性纤维化的主要病原体， 难以治疗，因为细菌形成了一个“生物膜群落”， 它们对传统抗生素不太敏感。群体感应，由 酰基高丝氨酸内酯（阿勒）信号传导分子，调节发病机制， 铜绿假单胞菌生物膜形成。因此，了解分子 群体感应的基础是一个高度优先的发展， 抗菌剂。该项目的长期目标是扩大 对群体感应系统的理解，以原子水平，以开发一个 控制细菌致病机制的详细描述。 该提案的主要焦点是产生阿勒的酶类 信号，AHL-激酶，因为缺乏阿勒信号的细菌不能成为 致病或形成稳定的生物膜。虽然有模型的机制 由于AHL酶的作用，目前还没有任何阿勒的结构 合成酶高分辨率的结构信息是绝对必要的 充分了解AHL的合成机理，为进一步研究AHL的合成提供依据 为未来的结构为基础的抑制剂设计开发新的 治疗学 本项目的具体目标是：（一）确定高分辨率 晶体结构的Pantoea stewartii subsp. Stewartii AHL-合酶（EsaI） 了解其功能，机制以及与其他酶的关系， 使用相似的基质。进行诱变、结合和动力学 用EsaI进行实验，以更好地理解催化机制， 底物特异性(II)研究铜绿假单胞菌AHL-合成酶，LasI，使用 结构和生化技术来了解阿勒的特异性 生产是确定的。(III)确定AHL合酶同源物 在趋异生物体中使用质量产生高丝氨酸内酯信号 光谱法和活性测定。研究结构和机制， 确定与其他阿勒酶的相似性。

DESCRIPTION (provided by applicant): Persistent bacterial infections are a major cause of death in cystic fibrosis patients and immune-compromised individuals. A number of gram-negative bacteria including Pseudomonas aeruginosa, a major pathogen in cystic fibrosis, cause infections that are difficult to treat because the bacteria form a "biofilm community" that renders them less sensitive to traditional antibiotics. Quorum sensing, mediated by acylhomoserine lactone (AHL) signaling molecules, regulates pathogenesis and biofilm formation in P. aeruginosa. Therefore, understanding the molecular basis of quorum sensing is a high priority in the development of novel anti-bacterial agents. The long term goal of this project is to extend the understanding of the quorum-sensing system to the atomic level to develop a detailed description of the mechanisms that control bacterial pathogenesis. The main focus of this proposal is the class of enzymes that produce the AHL signal, AHL-synthases, because bacteria lacking the AHL signal fail to become pathogenic or form stable biofilms. Although there are models of the mechanism of action of the AHL-synthases, there are currently no structures of any AHL synthase. High resolution structural information is absolutely essential for fully understanding the mechanism of AHL synthesis and will provide the basis for future structure-based inhibitor design for development of novel therapeutics. The specific aims for this project are: (I) determine the high resolution crystal structure of the Pantoea stewartii subsp. Stewartii AHL-synthase (EsaI) to understand its function, mechanism, and relationship to other enzymes that utilize similar substrates. Perform mutagenesis, binding and kinetics experiments with EsaI to better understand the catalytic mechanism and substrate specificity. (II) Study the P. aeruginosa AHL-synthase, LasI, using structural and biochemical techniques to understand how specificity of AHL production is determined. (III) Establish whether the AHL-synthase homologues in divergent organisms produce a homoserine lactone signal using mass spectrometry and activity assays. Study the structures and mechanisms to determine similarities to other AHL synthases.