The structure of Escherichia coli nitroreductase complexed with nicotinic acid: Three crystal forms at 1.7 (A)over-circle, 1.8 (A)over-circle and 2.4 (A)over-circle resolution
The structure of Escherichia coli nitroreductase complexed with nicotinic acid: Three crystal forms at 1.7 (A)over-circle, 1.8 (A)over-circle and 2.4 (A)over-circle resolution
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DOI：
10.1006/jmbi.2001.4653
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发表时间：
2001-05-25
影响因子：
5.6
通讯作者：
White, SA
中科院分区：
文献类型：
--
作者：
Lovering, AL;Hyde, EI;White, SA
大肠杆菌硝基还原酶是一种黄素蛋白，可还原多种醌和硝基芳香底物。它能够将相对无毒的前药如CB 1954（5-（aziridin-1-yl）-2，4-dinitrobenzamide）转化为高度细胞毒性的衍生物，这引起了人们对其用于癌症基因治疗的潜力的兴趣。我们已经确定了结合到底物类似物，烟酸，从三种晶体形式的分辨率为1.7埃，1.8埃和2.4埃代表10个非晶体学相关的单体的酶的结构。该酶是二聚体，并且具有大的疏水核心;分子的每一半由被α-螺旋包围的五链β-折叠组成。螺旋E和F从每个单体的核心区域突出。存在广泛的二聚体界面，并且15个C-末端残基围绕相对单体延伸，形成第五条β-链。活性位点位于分子的相对侧，在二聚体界面处的溶剂暴露的裂缝中。FMN形成氢键的一个单体和疏水性接触，它的硅面被掩埋。烟酸堆积在FMN的表面和螺旋F中的Phe 124之间，只有一个氢键与蛋白质结合。如果辅酶NAD（P）H的烟酰胺环与烟酸配体的烟酰胺环处于相同的位置，则其C4原子将被最佳地定位用于直接氢化物转移到黄素N5。与unliganded黄素还原酶和NTR的结构的比较表明，在配体结合时螺旋E和F的流动性降低。结构分析解释了酶的广泛底物特异性，并为合理设计新型前药和定点诱变以提高酶活性提供了基础。(C)北京：科学出版社.
Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 Angstrom, 1.8 Angstrom and 2.4 Angstrom representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta -sheet surrounded by alpha -helices. Helices E and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta -strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dlimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity. (C) 2001 Academic Press.