【作者】 姬小敏； 李欣； 何寅武； 牛聪伟； 沈月全； 席真；

【Author】 Xiaomin Ji;X. Li;Y. He;C. Niu;Y. Shen;Z. Xi;State Key Laboratory of Elemento-Organic Chemistry, University of Nankai;State Key Laboratory of Medicinal Chemical Biology, University of Nankai;

【机构】 南开大学化学学院元素有机化学国家重点实验室； 南开大学生命科学学院药物化学生物学国家重点实验室；

【摘要】 细菌、古菌、真菌和植物都具有支链氨基酸(缬氨酸、亮氨酸和异亮氨酸)的生物合成途径。乙酰羟酸合成酶(AHAS)是支链氨基酸生物合成途径中的第一个关键酶。几乎所有物种(古菌除外)的AHAS都含有两种亚基:催化亚基和调控亚基。亚基间的相互作用对酶行使功能至关重要。迄今为止,对于AHAS的催化反应机制、调控亚基对催化亚基的激活机制以及支链氨基酸对全酶活性的反馈调控机制知之甚少。因此获得AHAS全酶的晶体结构对解释AHAS的这三种机制无疑是非常有帮助的。自2002年以来,AHAS催化亚基和调控亚基的晶体结构先后被解析,然而AHAS全酶的晶体结构因其稳定性等原因,未能成功解析。我们在结构及序列分析的基础上,通过AHAS催化亚基柔性片段的刚性替换,以及在蛋白的端基引入重金属结合标签(dLBT)两种方式,构建AHAS突变体蛋白,以提高AHAS酶的稳定性,为获得AHAS全酶晶体结构进行了有益的探索,并取得了初步结果。更多还原

【Abstract】 Acetohydroxyacid synthase(AHAS, EC 2.2.1.6) catalyzes the first common step in the biosynthesis of branched chain amino acids. Most of the enzymes from various species is composed of catalytic and regulatory subunits. So far the mechanisms of catalysis, activation and feedback inhibition for AHAS were poorly understood. The study on the structure of AHAS is very important to explain these mechanisms. The crystal structures of AHAS catalytic and regulatory subunits have been determined by X-ray diffraction, respectively. However, the crystal structure of the holoenzyme has not been reported yet because of the difficulty in protein crystallography, such as stabilization of protein, growth of protein crystals, and so on. Herein, AHAS sequences and structures from various species were analysed, and the flexible segments of residues were identified. On the basis of that, the mutants of AHAS were constructed through replacement of the flexible segments and a double lanthanide-binding tag(dLBT) in order to increase the stabilization of protein and to aid the structure solution.更多还原