Ramón-Maiques Santiago, Fernández-Murga María Leonor, Gil-Ortiz Fernando, Vagin Alexei, Fita Ignacio, Rubio Vicente
Instituto de Biomedicina de Valencia (CSIC), Jaume Roig 11, Valencia 46010, Spain.
J Mol Biol. 2006 Feb 24;356(3):695-713. doi: 10.1016/j.jmb.2005.11.079. Epub 2005 Dec 12.
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 PII. 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 A and 2.95 A 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 approximately 30 A diameter. They are formed by linking three E.coli NAGK-like homodimers through the interlacing of an N-terminal mobile kinked alpha-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 approximately 4), and appears essential for arginine inhibition and for NAGK-PII complex formation, since this complex may involve binding of NAGK and PII 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.
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反应。在许多生物体中，该酶受到该途径最终产物精氨酸的抑制，因此发挥着核心调节作用。此外，在光合生物中，NAGK是氮信号蛋白PII的作用靶点。同源二聚体、对精氨酸不敏感的大肠杆菌NAGK的三维结构阐明了底物结合和催化过程，但未揭示精氨酸对NAGK的抑制作用机制。我们现在通过分别测定与精氨酸结合的嗜热栖热菌NAGK和无精氨酸的铜绿假单胞菌NAGK的晶体结构（分辨率分别为2.75 Å和2.95 Å），揭示了精氨酸抑制作用的机制。这两种酶均为高度相似的环状六聚体，具有直径约为30 Å的中心孔。它们是通过N端可移动的扭结α-螺旋相互交织，将三个类似大肠杆菌NAGK的同源二聚体连接而成，而大肠杆菌NAGK中不存在这种α-螺旋。精氨酸结合在嗜热栖热菌NAGK每个亚基中，位于二聚体间连接部位两侧，在亚基的N螺旋和C叶之间形成的位点上。该位点在铜绿假单胞菌NAGK中也存在，但构象不同，而在大肠杆菌NAGK中则不存在。精氨酸通过将每个亚基的C叶与二聚体间连接部位相连，可能稳定了扩大的活性中心构象，从而阻碍了催化作用。乙酰谷氨酸通过促进活性中心闭合来对抗精氨酸的抑制作用。六聚体结构解释了所观察到的具有高希尔系数（N约为4）的S形精氨酸抑制动力学，并且似乎对于精氨酸抑制作用和NAGK-PII复合物形成至关重要，因为该复合物可能涉及NAGK和PII以其三重轴对齐的方式结合。NAGK的结构使得能够识别出精氨酸抑制作用的诊断性序列特征。这些特征在同源的受精氨酸抑制的酶NAG合酶中也能找到。关于NAGK的研究结果揭示了该合酶的结构、功能和精氨酸抑制作用机制，并构建了一个六聚体模型。
