Ferreira Frederico M, Mendoza-Hernandez Guillermo, Castañeda-Bueno Maria, Aparicio Ricardo, Fischer Hannes, Calcagno Mario L, Oliva Glaucius
Instituto de Física de São Carlos, Universidade de São Paulo, C.P. 369, 13560-970 São Carlos, SP, Brazil.
J Mol Biol. 2006 Jun 2;359(2):308-21. doi: 10.1016/j.jmb.2006.03.024. Epub 2006 Mar 29.
We report the crystal structure of the apoenzyme of N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase from Escherichia coli (EcNAGPase) and the spectrometric evidence of the presence of Zn2+ in the native protein. The GlcNAc6P deacetylase is an enzyme of the amino sugar catabolic pathway that catalyzes the conversion of the GlcNAc6P into glucosamine 6-phosphate (GlcN6P). The crystal structure was phased by the single isomorphous replacement with anomalous scattering (SIRAS) method using low-resolution (2.9 A) iodine anomalous scattering and it was refined against a native dataset up to 2.0 A resolution. The structure is similar to two other NAGPases whose structures are known from Thermotoga maritima (TmNAGPase) and Bacillus subtilis (BsNAGPase); however, it shows a phosphate ion bound at the metal-binding site. Compared to these previous structures, the apoenzyme shows extensive conformational changes in two loops adjacent to the active site. The E. coli enzyme is a tetramer and its dimer-dimer interface was analyzed. The tetrameric structure was confirmed in solution by small-angle X-ray scattering data. Although no metal ions were detected in the present structure, experiments of photon-induced X-ray emission (PIXE) spectra and of inductively coupled plasma emission spectroscopy (ICP-AES) with enzyme that was neither exposed to chelating agents nor metal ions during purification, revealed the presence of 1.4 atoms of Zn per polypeptide chain. Enzyme inactivation by metal-sequestering agents and subsequent reactivation by the addition of several divalent cations, demonstrate the role of metal ions in EcNAGPase structure and catalysis.
我们报道了来自大肠杆菌的N-乙酰葡糖胺-6-磷酸（GlcNAc6P）脱乙酰酶（EcNAGPase）的脱辅基酶晶体结构，以及天然蛋白质中存在Zn2+的光谱学证据。GlcNAc6P脱乙酰酶是氨基糖分解代谢途径中的一种酶，催化GlcNAc6P转化为葡糖胺6-磷酸（GlcN6P）。晶体结构通过使用低分辨率（2.9 Å）碘反常散射的单同晶置换加反常散射（SIRAS）方法进行相位确定，并针对高达2.0 Å分辨率的天然数据集进行精修。该结构与另外两种已知结构的NAGPases相似，分别来自嗜热栖热菌（TmNAGPase）和枯草芽孢杆菌（BsNAGPase）；然而，它在金属结合位点处结合有一个磷酸根离子。与这些先前的结构相比，脱辅基酶在与活性位点相邻的两个环中显示出广泛的构象变化。大肠杆菌酶是一种四聚体，并对其二聚体-二聚体界面进行了分析。通过小角X射线散射数据在溶液中证实了四聚体结构。尽管在当前结构中未检测到金属离子，但对在纯化过程中既未暴露于螯合剂也未暴露于金属离子的酶进行的光子诱导X射线发射（PIXE）光谱和电感耦合等离子体发射光谱（ICP-AES）实验表明，每条多肽链存在1.4个Zn原子。金属螯合剂使酶失活，随后添加几种二价阳离子使其重新激活，这证明了金属离子在EcNAGPase结构和催化中的作用。
