Sub-atomic resolution crystal structure of cholesterol oxidase: What atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity
Sub-atomic resolution crystal structure of cholesterol oxidase: What atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity
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DOI：
10.1016/s0022-2836(03)00054-8
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发表时间：
2003-03-07
影响因子：
5.6
通讯作者：
Vrielink, A
中科院分区：
文献类型：
--
作者：
Lario, PI;Sampson, N;Vrielink, A
将胆固醇氧化酶（一种56kDa黄素酶）的晶体结构各向异性地精修至0.95埃分辨率。最终的晶体学R因子和无R值分别为11.0%和13.2%。电子密度图的质量，使替代构象的酶中的83个残基，其中许多位于活性位点的建模。在先前的高分辨率结构（1.5埃分辨率）中观察到的其他结构特征并不明显，并且能够识别直接通向FAD辅基的异咯嗪部分的狭窄通道。这种狭窄通道的疏水性质表明它是分子氧进入异咯嗪基团进行氧化半反应的途径。解析活性位点残基中的交替构象为底物结合的动力学和涉及进入疏水隧道的潜在氧化触发门控机制提供了模型。这种结构表明，NE2原子的活性，网站组氨酸残基，H447，这黄素氧化酶的氧化还原活性的关键，作为一个氢键供体，而不是作为氢受体。胆固醇氧化酶的原子分辨率结构揭示了氢原子的存在，蛋白质的动力学方面以及侧链构象如何与新的结构特征（如氧通道）相关。这些新的结构信息为我们提供了重新分析特定残基在酶机制中所起作用的机会。(C)2003爱思唯尔科技有限公司版权所有。
The crystal structure of cholesterol oxidase, a 56 kDa flavoenzyme was anisotropically refined to 0.95 Angstrom resolution. The final crystallographic R-factor and R-free value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5 Angstrom resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active, site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme. (C) 2003 Elsevier Science Ltd. All rights reserved.