Mechanism and Inhibition of S-Ribosylhomocysteinase

描述（由申请人提供）：群体感应调节许多细菌行为，包括毒力、生物膜形成、毒素和抗生素产生、发光和运动性。不同种类的细菌使用呋喃糖基衍生物作为2型自诱导物（AI-2）用于种间通信。AI-2由S-腺苷甲硫氨酸通过三个酶促步骤生物合成。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，以及（2）开发针对AI-2合成和/或检测的特异性抑制剂作为新型抗菌剂。在此资助期间，该项目将重点研究S-核糖基高半胱氨酸酶（LuxS）的催化机制和抑制作用，该酶催化AI-2生物合成的最后一步。LuxS是一种新型的不含亚铁血红素的酶，具有新颖的催化机制。在具体目标1中，将化学合成推定的催化中间体和底物类似物，并进行动力学表征，以证明其参与催化途径。将通过X射线晶体学确定与这些中间体和类似物复合的LuxS的结构。具体目标2是通过电子吸收和磁性圆二色光谱确定金属离子在催化中的作用。具体目标3是通过定点诱变和突变体的动力学和光谱表征来确定保守活性位点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的金属螯合抑制剂。将检测抑制剂对LuxS的抑制和抗菌活性。基于机制的抑制剂也将作为研究LuxS机制的机制探针。最后，具体目标5是纯化和鉴定AI-2的其他受体。

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.