Comparative structural and functional analysis of Pseudomonas aeruginosa inhibitor of vertebrate lysozyme paralogs

项目总结-铜绿假单胞菌抑制剂的结构和功能比较分析 脊椎动物的溶菌酶。 托马斯·利珀，PI，肯纳索州立大学。 拟议的综合研究将调查肽聚糖生物化学的一个重要调节因素。 囊性纤维化病原菌铜绿假单胞菌(PA)，已知显示多药耐药 (MDR)。具体地说，脊椎动物溶菌酶蛋白2抑制物(IVYp2)的结构将得到确定 通过核磁共振(Aim 1)和比较其抑制水解酶动力学的能力(Aim 2)。而艾薇 蛋白质最初被认为是抑制溶菌酶糖苷水解酶的，后来的工作表明 常春藤的进化起源可能是抑制了裂解转糖基酶(LTS)。例如, IVYp2在酶抑制试验中对溶菌酶活性没有影响，但已被证明抑制LTS， 至少在体外是这样。为了确定哪些LT是体内可识别的，用酶抑制试验 LTS(目标2)将与互补和遗传编码的新应用进行比较 常春藤基因敲除PA菌株的交联研究(目标3)。确定的同源常春藤-LT对将是 与IVYp2(目标1)的核磁共振结构和其他IVY2的现有复杂结构(即 IVYp1)与溶菌酶结合，提示LT分子识别的重要区域。毕业生和 本科生，特别是那些来自代表人数不足的群体的学生，将显著参与 这项研究促进了数据收集，同时为他们提供了重要的培训和进步 机遇。这些创新的研究将建立未来所需的背景和工作流程 药物发现努力开发针对MDR PA中新的生化途径的新药物类别。

Project Summary – Comparative structural and functional analysis of Pseudomonas aeruginosa inhibitor of vertebrate lysozyme paralogs. Thomas Leeper, PI, Kennesaw State University. The proposed integrative studies will investigate a significant regulator of peptidoglycan biochemistry in the cystic fibrosis pathogen Pseudomonas aeruginosa (PA), known to display multidrug resistance (MDR). Specifically, Inhibitor of Vertebrate Lysozyme protein 2 (IVYp2) will have its structure determined by NMR (Aim 1) and compared to its ability to inhibit hydrolase enzyme kinetics (Aim 2). While IVY proteins were originally suggested to inhibit lysozyme glycoside hydrolases, subsequent work suggested that the evolutionary origin of IVY’s may have been to inhibit lytic transglycosylases (LTs). For example, IVYp2 has no effect on lysozyme activity in enzyme inhibition assays but has been shown to inhibit LTs, at least in vitro. In order to determine which LTs are recognized in vivo enzyme inhibition assays with LTs (Aim 2) will be compared to novel applications of complementation and genetically encoded crosslinking studies in IVY knockout PA strains (Aim 3). The identified cognate IVY-LT pairs will be compared to the NMR structure of IVYp2 (Aim 1) and existing complex structures for other IVYs (i.e. IVYp1) bound to lysozyme to suggest regions important for LT molecular recognition. Graduate and undergraduate students, particularly those from underrepresented groups, will significantly participate in this research to facilitate data collection while providing them important training and advancement opportunities. These innovative studies will establish the background and workflow needed for future drug discovery efforts to develop new drug classes targeting novel biochemical pathways in MDR PA.