Structural bases of feed-back control of arginine biosynthesis, revealed by the structures of two hexameric N-acetylglutamate kinases, from Thermotoga maritima and Pseudomonas aeruginosa
Structural bases of feed-back control of arginine biosynthesis, revealed by the structures of two hexameric N-acetylglutamate kinases, from Thermotoga maritima and Pseudomonas aeruginosa
复制标题
DOI：
10.1016/j.jmb.2005.11.079
复制
发表时间：
2006-02-24
影响因子：
5.6
通讯作者：
Rubio, V
中科院分区：
文献类型：
--
作者：
Ramón-Maiques, S;Fernández-Murga, ML;Rubio, V
N-乙酰谷氨酸激酶（NAGK）催化精氨酸生物合成途径中的第二步。在许多生物体中，这种酶被该途径的最终产物精氨酸抑制，因此起着中心调节作用。此外，在光合生物中，NAGK是氮信号蛋白PIT的靶标。同源二聚体，精氨酸不敏感，大肠杆菌NAGK的3-D结构，澄清底物结合和催化，但没有揭示精氨酸抑制NAGK。我们现在通过分别在2.75埃和2.95埃分辨率下确定精氨酸复合的海栖热袍菌和无精氨酸的铜绿假单胞菌NAGK的晶体结构来阐明精氨酸抑制。两种酶都是高度相似的环状六聚体，具有类似于30埃直径的中心孔。它们由三个E连接而成。coliNAGK样同源二聚体通过N-末端移动的扭结的α-螺旋的交错而形成，这在E. coli NAGK。精氨酸结合在T的每个亚基中。maritima NAGK，位于二聚体间连接的侧翼，在亚基的N螺旋和C叶之间形成的位点。该位点也以不同的构象存在于铜绿假单胞菌NAGK中，但在大肠杆菌中缺失。coli NAGK。精氨酸通过将每个亚基的C端与二聚体间连接处粘合，可以稳定扩大的活性中心构象，从而阻碍催化。乙酰谷氨酸通过促进活性中心关闭来对抗精氨酸抑制。六聚体结构证明观察到的S形精氨酸抑制动力学具有高希尔系数（N近似为4），并且对于精氨酸抑制和NAGK-P-II复合物形成似乎是必需的，因为该复合物可能涉及NAGK和PIT的结合，其3倍轴对齐。NAGK结构允许鉴定精氨酸抑制的诊断序列特征。这些特征也在同源的精氨酸抑制酶NAG合酶中发现。NAGK的研究结果揭示了这种合成酶的结构，功能和精氨酸抑制，为此构建了一个六聚体模型。(c)2005爱思唯尔有限公司保留所有权利。
N-Acetylglutamate kinase (NAGK) catalyses the second step in the route of arginine biosynthesis. In many organisms this enzyme is inhibited by the final product of the route, arginine, and thus plays a central regulatory role. In addition, in photosynthetic organisms NAGK is the target of the nitrogen-signalling protein PIT. The 3-D structure of homodimeric, arginine-insensitive, Escherichia coli NAGK, clarified substrate binding and catalysis but shed no light on arginine inhibition of NAGK. We now shed light on arginine inhibition by determining the crystal structures, at 2.75 angstrom and 2.95 angstrom resolution, of arginine-complexed Thermotoga maritima and arginine-free Pseudomonas aeruginosa NAGKs, respectively. Both enzymes are highly similar ring-like hexamers having a central orifice of similar to 30 angstrom diameter. They are formed by linking three E. coli NAGK-like homodimers through the interlacing of an N-terminal mobile kinked a-helix, which is absent from E. coli NAGK. Arginine is bound in each subunit of T. maritima NAGK, flanking the interdimeric junction, in a site formed between the N helix and the C lobe of the subunit. This site is also present, in variable conformations, in P. aeruginosa NAGK, but is missing from E. coli NAGK. Arginine, by gluing the C lobe of each subunit to the inter-dimeric junction, may stabilize an enlarged active centre conformation, hampering catalysis. Acetylglutamate counters arginine inhibition by promoting active centre closure. The hexameric architecture justifies the observed sigmoidal arginine inhibition kinetics with a high Hill coefficient (N approximate to 4), and appears essential for arginine inhibition and for NAGK-P-II complex formation, since this complex may involve binding of NAGK and PIT with their 3-fold axes aligned. The NAGK structures allow identification of diagnostic sequence signatures for arginine inhibition. These signatures are found also in the homologous arginine-inhibited enzyme NAG synthase. The findings on NAGK shed light on the structure, function and arginine inhibition of this synthase, for which a hexameric model is constructed. (c) 2005 Elsevier Ltd. All rights reserved.