Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism
Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism
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AzrA 和 AzrC 与底物或抑制剂复合的结构：深入了解底物特异性和催化机制
DOI：
10.1107/s1399004713030988
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发表时间：
2014-02-01
影响因子：
2.2
通讯作者：
Yao, Min
中科院分区：
文献类型：
--
作者：
Yu, Jian;Ogata, Daiki;Yao, Min
偶氮染料是一种主要的合成染料，具有一个或多个偶氮键，广泛应用于各种工业用途。偶氮还原酶催化降解偶氮染料废水是一种非常有效的废水处理方法。因此，偶氮还原酶与各种底物的复合物的结构对于理解其底物特异性和催化机制是必不可少的。本研究测定了偶氮染料酸性红88（AR 88）和橙子I（OI）与汽巴蓝（CB）络合的AzrA和AzrC的晶体结构。CB作为NAD（P）H的抑制剂/类似物，位于黄素单核苷酸（FMN）的顶部，表明CB与NAD（P）H以类似的方式直接结合到FMN上。AzrC-AR 88和AzrC-OI复合物的结构分别显示了两种方式与偶氮键邻位或帕拉具有羟基的底物结合，而据ITC实验估计，AR 88和OI与AzrC具有相似的结合亲和力。虽然这两个基板结合在不同的方向，羟基基团位于相似的位置，导致亲电子C原子结合的质子/电子供体的距离类似于3.5埃的FMN的N5的安排。根据晶体结构和定点突变分析，提出了不同底物的催化机制。
Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P) H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P) H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of similar to 3.5 angstrom to N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.