Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, Sichuan Key Laboratory of Molecular Biology and Biotechnology, College of Life Sciences, Sichuan University, Chengdu, Sichuan, P. R. China.
J Biochem. 2011 Jun;149(6):673-84. doi: 10.1093/jb/mvr015. Epub 2011 Feb 3.
Three-dimensional structural model of epoxide hydrolase (PchEHA) from Phanerochaete chrysosporium was constructed based on X-ray structure of Agrobacterium radiobacter AD1 sEH using SWISS-MODEL server. Conserved residues constituting the active site cavity were identified, of which the functional roles of 14 residues were determined by site-directed mutagenesis. In catalytic triad, Asp105 and His308 play a leading role in alkylation and hydrolysis steps, respectively. Distance between Asp105 and epoxide ring of substrate may determine the regiospecificity in the substrate docking model. Asp277 located at the entrance of substrate tunnel is concerned with catalysis but not essential. D307E had the highest activity and lower enantioselectivity among 14 mutants, suggesting Asp307 may be involved in choice of substrate configuration. Y159F and Y241F almost exhibited no activity, indicating that they are essential to bind substrate and facilitate opening of epoxide ring. Besides, His35-Gly36-Asn37-Pro38, Trp106 and Trp309 surrounding Asp105, may coordinate the integration of active site cavity and influence substrate binding. Especially, W106I reversed the enantioselectivity, perhaps due to more deteriorative impact on the preferred (R)-styrene oxide. Gly65 and Gly67 occurring at β-turns and Gly36 are vital in holding protein conformation. Conclusively, single conserved residue around the active sites has an important impact on catalytic properties.
利用 SWISS-MODEL 服务器，根据黄孢原毛平革菌（Phanerochaete chrysosporium）环氧化物水解酶（PchEHA）的 X 射线结构，构建了该酶的三维结构模型。鉴定了构成活性位点腔的保守残基，通过定点突变确定了其中 14 个残基的功能作用。在催化三联体中，Asp105 和 His308 分别在烷基化和水解步骤中起主导作用。Asp105 与底物环氧环之间的距离可能决定了在底物对接模型中立体特异性。位于底物隧道入口处的 Asp277 与催化有关，但不是必需的。在 14 个突变体中，D307E 的活性最高，对映选择性最低，表明 Asp307 可能参与了底物构型的选择。Y159F 和 Y241F 几乎没有活性，表明它们对于结合底物和促进环氧环的打开是必需的。此外，环绕 Asp105 的 His35-Gly36-Asn37-Pro38、Trp106 和 Trp309 可能协调活性位点腔的整合并影响底物结合。特别是，W106I 反转了对映选择性，可能是因为对优选的（R）-苯乙烯氧化物的影响更具破坏性。发生在β-转角和 Gly36 处的 Gly65 和 Gly67 对于保持蛋白质构象至关重要。总之，活性位点周围的单个保守残基对催化特性有重要影响。
