Mechanism and Inhibition of S-Ribosylhomocysteinase

描述(由申请人提供)：群体感应调节一系列细菌行为，包括毒力、生物膜形成、毒素和抗生素的产生、发光和运动。不同种类的细菌使用呋喃糖衍生物作为2型自动诱导剂(AI-2)进行物种间交流。AI-2是以S-腺苷蛋氨酸为原料，经三步酶促合成的。该项目的长期目标是(1)确定参与细菌群体感应的酶的催化机制，(2)开发针对AI-2作为新型抗菌剂的合成和/或检测的特定抑制剂。在此资助期间，该项目将重点研究催化AI-2生物合成最后一步的S-核糖基同型半胱氨酸酶(LUXS)的催化机理和抑制。LuxS是一种新型的非血红素Fe2+酶，具有新的催化机理。在具体目标1中，假定的催化中间体和底物类似物将被化学合成并进行动力学表征，以证明它们参与了催化途径。这些中间体和类似物与LuxS形成的络合物的结构将由X-射线结晶学确定。具体目标2是用电子吸收光谱和磁圆二色谱确定金属离子在催化中的作用。具体目标3是通过定点突变以及突变体的动力学和光谱表征来确定保守的活性中心残基在催化中的功能。在具体目标4中，将设计和合成基于机理和金属螯合的缓蚀剂。将测试这些抑制剂对LuxS的抑制作用和抗菌活性。这些以机制为基础的抑制剂也将作为研究LuxS机制的机制探针。最后，特异靶5是纯化和鉴定AI-2的额外受体(S)。

DESCRIPTION (provided by applicant): Quorum sensing regulates a host of bacterial behaviors including virulence, biofilm formation, toxin and antibiotic production, luminescence, and motility. Different species of bacteria use a furanosyl derivative as the type 2 autoinducer (AI-2) for interspecies communication. AI-2 is biosynthesized from S-adenosylmethionine via three enzymatic steps. The long-term objectives of this project are (1) to determine the catalytic mechanisms of enzymes involved in bacterial quorum sensing, and (2) to develop specific inhibitors against the synthesis and/or detection of AI-2 as novel antibacterial agents. During this grant period, the project will focus on the catalytic mechanism and inhibition of S-ribosylhomocysteinase (LuxS), which catalyzes the last step of AI-2 biosynthesis. LuxS is a new type of non-heme Fe2+containing enzyme and has a novel catalytic mechanism. In Specific Aim 1, putative catalytic intermediates and substrate analogs will be chemically synthesized and kinetically characterized to demonstrate their involvement in the catalytic pathway. The structures of LuxS in complex with these intermediates and analogs will be determined by X-ray crystallography. Specific Aim 2 is to determine the role of the metal ion in catalysis by electronic absorption and magnetic circular dichroism spectroscopies. Specific Aim 3 is to determine the function of conserved active-site residues in catalysis by site-directed mutagenesis and kinetic and spectroscopic characterization of the mutants. In Specific Aim 4, mechanism-based and metal-chelating inhibitors will be designed and synthesized. The inhibitors will be tested for inhibition of LuxS and antibacterial activity. The mechanism-based inhibitors will also serve as mechanistic probes for studying the LuxS mechanism. Finally, Specific Aim 5 is to purify and identify additional receptor(s) for AI-2.