Cyanuric acid hydrolase from Azorhizobium caulinodans ORS 571: crystal structure and insights into a new class of Ser-Lys dyad proteins.
Cyanuric acid hydrolase from Azorhizobium caulinodans ORS 571: crystal structure and insights into a new class of Ser-Lys dyad proteins.
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DOI：
10.1371/journal.pone.0099349
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Aihara H
中科院分区：
文献类型：
--
作者：
Cho S;Shi K;Seffernick JL;Dodge AG;Wackett LP;Aihara H
氰尿酸水解酶（CAH）催化氰尿酸（2，4，6-三羟基-1，3，5-三嗪）的水解开环，氰尿酸是均三嗪细菌降解的中间产物，也是三氯异氰尿酸消毒的副产物。在本研究中，一个CAH-巴比妥酸抑制剂复合物Azorhizobium caulinodans ORS 571的X-射线晶体结构已被确定在2.7 μ m分辨率。CAH蛋白折叠由三个结构上同源的结构域组成，形成β-桶样结构，具有外部α-螺旋，导致三重对称，这是结构和活性位点的主要特征，反映了底物氰尿酸的三重对称形状。CAH的活性位点结构与最近确定的AtzD相似，具有三对活性位点Ser-Lys二联体。为了确定每个Ser-Lys二联体在催化中的作用，使用高灵敏度的酶偶联测定进行了突变研究。S226 A突变体的109倍活性损失比S79 A和S333 A突变体低至少10倍。此外，生物信息学分析显示Ser 226/Lys 156二联体是CAH/巴比妥酶家族中唯一绝对保守的二联体。这些数据表明，Lys 156激活Ser 226亲核试剂，然后可以攻击底物羰基。我们的结构，突变和生物信息学分析相结合，区分这项研究，并提供实验数据的机制洞察到这个独特的蛋白质家族。
Cyanuric acid hydrolase (CAH) catalyzes the hydrolytic ring-opening of cyanuric acid (2,4,6-trihydroxy-1,3,5-triazine), an intermediate in s-triazine bacterial degradation and a by-product from disinfection with trichloroisocyanuric acid. In the present study, an X-ray crystal structure of the CAH-barbituric acid inhibitor complex from Azorhizobium caulinodans ORS 571 has been determined at 2.7 Å resolution. The CAH protein fold consists of three structurally homologous domains forming a β-barrel-like structure with external α-helices that result in a three-fold symmetry, a dominant feature of the structure and active site that mirrors the three-fold symmetrical shape of the substrate cyanuric acid. The active site structure of CAH is similar to that of the recently determined AtzD with three pairs of active site Ser-Lys dyads. In order to determine the role of each Ser-Lys dyad in catalysis, a mutational study using a highly sensitive, enzyme-coupled assay was conducted. The 109-fold loss of activity by the S226A mutant was at least ten times lower than that of the S79A and S333A mutants. In addition, bioinformatics analysis revealed the Ser226/Lys156 dyad as the only absolutely conserved dyad in the CAH/barbiturase family. These data suggest that Lys156 activates the Ser226 nucleophile which can then attack the substrate carbonyl. Our combination of structural, mutational, and bioinformatics analyses differentiates this study and provides experimental data for mechanistic insights into this unique protein family.
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