微生物乳酸代谢调控蛋白LldR的结构及机制研究

项目名称： 微生物乳酸代谢调控蛋白LldR的结构及机制研究

项目编号： No.31270090

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 生物科学

项目作者： 马翠卿

作者单位： 山东大学

项目金额： 82万元

中文摘要： 致病菌乳酸代谢在其抵抗人体免疫系统攻击、感染人体过程中起重要作用。课题组前期从事假单胞菌乳酸代谢机制研究，成功实现了条件致病菌铜绿假单胞菌XMG中乳酸代谢调控蛋白LldR的基因克隆，外源表达与生物学功能鉴定。LldR属于转录调控蛋白中的FadR家族，该家族蛋白调控微生物丙酮酸、乳酸、乙醇酸以及脂肪酸代谢等一系列重要生理代谢过程。前期研究表明，LldR与已报道的FadR在结构与调控机制方面可能存在明显区别。本申请拟以XMG中的LldR为研究对象，基于蛋白质结晶与X-射线衍射技术，解析LldR的晶体结构；采取定点突变、凝胶阻滞、DNA水解酶I足迹分析等技术确定LldR与DNA结合的关键氨基酸位点；利用等温滴定量热、表面等离子共振、圆二色谱等技术，阐明XMG中LldR调控该菌株乳酸代谢的具体机制；为深入揭示该家族蛋白调控的重要微生物代谢过程奠定理论基础。

中文关键词： 假单胞菌；乳酸分解代谢；调控蛋白；调控机制；生物催化

英文摘要： Lactate metabolism of pathogenic bacteria plays an important role in their resistance to the body's immune system and infection with the human. The lactate metabolism mechanism in different Pseudomonas strains was studied in our previous works. The lactate metabolism regulator LldR in the opportunistic pathogen Pseudomonas aeruginosa XMG was cloned, exogenous expressed and its biological function was successfully identified. LldR belongs to the transcriptional regulatory protein FadR family. The proteins in this family regulate a series of important physiological and metabolic processes including pyruvic acid, lactic acid, glycolic acid and fatty acid metabolism of bacteria. Preliminary studies show that there might have significant differences in the structure and regulatory mechanism between the LldR and the well studied FadR. In this study, the crystal structure of LldR in P. aeruginosa XMG would be analyzed by protein crystallization and X-ray diffraction techniques. Using site-directed mutagenesis, electrophoretic mobility shift assay and DNA hydrolase I footprinting analysis, the key amino acids sites in the binding of LldR to DNA would be determined. Using isothermal titration calorimetry, surface plasmon resonance, and circular dichroism, the regulation mechanisms of LldR in P. aeruginosa XMG would be cl

英文关键词： Pseudomonas；lactate catabolism；regulator；regulation mechanism；biocatalysis