钝齿棒杆菌精氨酸合成关键酶N-乙酰谷氨酸激酶晶体结构及其反馈抑制调节机制解析

L-Arginine biosynthesis pathway and metabolic regulation mechanisms are being studied as the typical biosynthesis regulations of the amino acids. In our previous research, excellent achievements of metabolic engineering were carried out in Corynebacterium crenatum SYPA, the hyper L-arginine producing strains were gained and its production level is the highest in our domestic field. The rational modifications with an aim for the resistant Ccre_NAGK to feedback-inhibition of L-arginine, the limiting-rate enzymatic reaction for L-arginine synthesis in Corynebacterium crenatum, were accomplished. A series of variant Ccre_NAGKs resistant to L-arginine were acquired (Amino Acids, 2012 43:255-266. IF 4.104). To gain insight into the structural and regulatory complexities of ascomycetal NAGKs, we carried out in vitro expression and crystallographic studies of Ccre_NAGK. In the present project, on the basis of getting the single crystal structure of Ccre_NAGK, the crystal and 3D structures of complex proteins of wild type, the mutant Ccre_NAGKs and their interactions with the substrate (NAG), ATP and the product (L-arginine) would be first elucidated and analyzed, respectively. At the same time, the catalytic mechanisms and the principles of the feedback-inhibition of wide-type Ccre_NAGK would be clarified as well. Consequently, the expected results would provide a theoretical principle for the metabolic regulation of the L-arginine biosynthesis. This could pave a way not only for the efficient synthesis of L-arginine, but also for the researches on mechanisms of the other feedback-inhibition in the amino acids biosynthesis pathway of Corynebacteria.

L-精氨酸的合成代谢调控机制作为氨基酸代谢调控的典型正被广泛研究。钝齿棒杆菌SYPA是课题组具有自主知识产权的一株高产精氨酸工业用菌株。研究组前期针对其精氨酸合成关键酶Ccre_NAGK进行了抗反馈抑制半理性改造，突变酶解除了受精氨酸的反馈抑制(Amino Acids, 2012 43:255-266. IF 4.106)。项目拟在已获得的Ccre_NAGK单晶基础上，测定并解析Ccre_NAGK及其突变体与底物、ATP和精氨酸等相互作用的复合蛋白质三维结构，从分子水平上探讨Ccre_NAGK和产物或金属离子特异性结合的结构基础，深入了解其反馈抑制调节及其变构催化原理，分析Ccre_NAGK-Arginine结合域，对该结合域多位点进行定点饱和突变，通过设置系列反馈抑制解除条件，阐明钝齿棒杆菌NAGK的变构反馈调节效应机制；为AAK家族酶抗反馈抑制提供理论基础。

L-精氨酸是一种重要的半必需氨基酸，广泛应用于食品、医药与化工领域。N-乙酰谷氨酸激酶(CcNAGK)是高产精氨酸菌株Corynebacterium crenatum SYPA5-5合成精氨酸途径中的关键酶，受到产物精氨酸的反馈抑制。首先对CcNAGK的精氨酸结合域的关键位点E19进行饱和突变，解析精氨酸对NAGK的结合机制；同时获得解除其反馈抑制的突变体E19Y。根据分子对接、CcNAGK底物结合位点结构分析，对活性位点G71、I74、F91进行饱和突变，通过动力学参数的测定，阐释酶活变化规律，为氨基酸激酶家族的催化机理的阐释提供了理论基础；同时也获得了提高CcNAGK的催化活性与稳定性的突变体I74V、F91H、F91Y。根据同源序列的比对，对α折叠与β折叠的转角处位点K234进行定点突变，来获得显著提高热稳定性的突变体K234T。为提高L-精氨酸的合成效率，将四个位点进行组合突变（E19Y/I74V/F91H/K234T）并引入钝齿棒杆菌中，构建重组菌C.crenatumSYPA-4，不仅解除了L-精氨酸的反馈抑制，且胞内NAGK酶活力与热稳定性均有显著提高；在5L发酵罐中重组菌的L-精氨酸产量61.2 g/L，产率为 0.635 g/l/h，比出发菌株（含野生型NAGK）提高了41.8％，精氨酸的合成速率显著提高。本研究解析了氨基酸激酶家族中NAGK的催化机理及反馈机理并在此基础上提升了CcNAGK的酶学属性，构建获得了既解除了精氨酸的反馈抑制作用又显著提高催化活性与热稳定性的CcNAGK突变体，提高了L-精氨酸产量和产率。