Biochemical, Spectroscopic and X-Ray Structural Analysis of Deuterated Multicopper Oxidase CueO Prepared from a New Expression Construct for Neutron Crystallography
Biochemical, Spectroscopic and X-Ray Structural Analysis of Deuterated Multicopper Oxidase CueO Prepared from a New Expression Construct for Neutron Crystallography
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由用于中子晶体学的新表达构建体制备的氘代多铜氧化酶 CueO 的生化、光谱和 X 射线结构分析
DOI：
10.1107/s2053230x1601400x
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Naoki Shibata
中科院分区：
文献类型：
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作者：
Mahfuza Akter;Chika Inoue;Hirofumi Komori;Nana Matsuda;Takeshi Sakurai;Kunishige Kataoka;Yoshiki Higuchi;Naoki Shibata
多铜氧化酶通过使用分子氧作为电子受体来氧化各种酚类和非酚类化合物以产生水。来自大肠杆菌的多铜氧化酶蛋白CueO参与细菌细胞中的铜稳态。虽然已经进行了X射线晶体学研究，但由于仅从X射线衍射数据难以识别H原子，因此氧的还原机制和质子转移途径仍然不清楚。为了阐明反应机制，使用中子晶体学，制备系统获得大，高品质的氘代CuO单晶的开发。从最初由原始构建体制备的氘代CueO获得微小晶体。氘代CueO的X-射线晶体结构表明，该蛋白在N-末端含有不完全截短的信号序列，这导致用于结晶的蛋白样品的异质性。在此，构建了一种新的CueO表达系统，该系统在信号序列之后具有HRV 3C切割位点。来自新构建体的氘代CueO在氘代藻类提取物培养基中培养的细胞中表达，并且信号序列被HRV 3C蛋白酶完全消除。通过MALDI-TOF质谱法估计纯化蛋白的氘代水平与非氘代蛋白相比至少为83.2%。非氘化CueO结晶空间群为P21，晶胞参数a = 49.51，B = 88.79，c = 53.95 Å，β = 94.24°，氘化CueO结晶空间群为P212121，晶胞参数a = 49.91，B = 106.92，c = 262.89 Å。 新结构晶体的晶体学参数与先前报道的非氘代晶体不同。从新的结构的非氘代和氘代CueO具有类似的紫外-可见光谱，酶活性和整体结构和几何形状的配体的Cu原子的活性位点的CueO结构。这些结果表明，CueO蛋白制备使用新的构建体是适合于进一步的中子衍射研究。
Multicopper oxidases oxidize various phenolic and nonphenolic compounds by using molecular oxygen as an electron acceptor to produce water. A multicopper oxidase protein, CueO, from Escherichia coli is involved in copper homeostasis in the bacterial cell. Although X-ray crystallographic studies have been conducted, the reduction mechanism of oxygen and the proton-transfer pathway remain unclear owing to the difficulty in identifying H atoms from X-ray diffraction data alone. To elucidate the reaction mechanism using neutron crystallography, a preparation system for obtaining large, high-quality single crystals of deuterated CueO was developed. Tiny crystals were obtained from the deuterated CueO initially prepared from the original construct. The X-ray crystal structure of the deuterated CueO showed that the protein contained an incompletely truncated signal sequence at the N-terminus, which resulted in the heterogeneity of the protein sample for crystallization. Here, a new CueO expression system that had an HRV3C cleavage site just after the signal sequence was constructed. Deuterated CueO from the new construct was expressed in cells cultured in deuterated algae-extract medium and the signal sequence was completely eliminated by HRV3C protease. The deuteration level of the purified protein was estimated by MALDI-TOF mass spectrometry to be at least 83.2% compared with nondeuterated protein. Nondeuterated CueO crystallized in space group P21, with unit-cell parameters a = 49.51, b = 88.79, c = 53.95 Å, β = 94.24°, and deuterated CueO crystallized in space group P212121, with unit-cell parameters a = 49.91, b = 106.92, c = 262.89 Å. The crystallographic parameters for the crystals of the new construct were different from those previously reported for nondeuterated crystals. The nondeuterated and deuterated CueO from the new construct had similar UV–Vis spectra, enzymatic activities and overall structure and geometry of the ligands of the Cu atoms in the active site to those of previously reported CueO structures. These results indicate that the CueO protein prepared using the new construct is suitable for further neutron diffraction studies.