Cyanuric Acid Hydrolase from Azorhizobium caulinodans ORS 571: Crystal Structure and Insights into a New Class of Ser-Lys Dyad Proteins

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.

氰尿酸水解酶（cyanuric acid hydrolase，CAH）可催化氰尿酸（2,4,6-三羟基-1,3,5-三嗪）的水解开环反应；氰尿酸是均三嗪（s-triazine）类化合物细菌降解过程中的中间产物，同时也是三氯异氰尿酸（trichloroisocyanuric acid）消毒过程中的副产物。本研究解析了来自茎瘤固氮根瘤菌（Azorhizobium caulinodans）ORS 571的CAH与巴比妥酸抑制剂复合物的X射线晶体结构（X-ray crystal structure），分辨率达2.7埃（Å）。CAH的蛋白质折叠由三个结构同源的结构域构成，形成带有外部α螺旋（α-helices）的类β桶状结构（β-barrel-like structure），整体呈现三重对称性（three-fold symmetry）；该对称性是该酶结构与活性中心（active site）的核心特征，与底物氰尿酸的三重对称结构高度契合。CAH的活性中心结构与近期解析的AtzD相似，二者均包含三对Ser-Lys二联体（Ser-Lys dyad）。为明确每一对Ser-Lys二联体在催化过程中的功能，本研究采用高灵敏度酶偶联检测法（enzyme-coupled assay）开展了突变体研究。S226A突变体的酶活性损失达109倍，其活性损失程度至少较S79A与S333A突变体低10倍。此外，生物信息学分析（bioinformatics analysis）显示，Ser226/Lys156二联体是CAH/巴比妥酶（barbiturase）家族中唯一完全保守的二联体。上述数据表明，Lys156可激活作为亲核试剂（nucleophile）的Ser226，使其能够攻击底物的羰基（carbonyl）位点。本研究整合结构解析、突变体分析与生物信息学分析手段，相较于同类研究形成了独特的研究维度，并为解析该独特蛋白质家族的催化机制提供了实验数据支撑。