MehpH的生化与结构特性

Biochemical and structural characterization of a monoethylhexyl phthalate hydrolase from Gordonia alkanivorans strain YC-RL2

摘要:

[目的]本研究旨在系统探究环境因素对邻苯二甲酸单乙基己基酯水解酶（monoethylhexyl phthalate hydrolase，MehpH）活性的影响，模拟该酶的3D结构并分析活性中心氨基酸残基与底物之间的相互作用。[方法]根据标准的酶学实验验证了环境因素对酶活性的影响。该酶的基本结构分析由DNAMAN （2.1）分析所得，同时利用SWISS-MODEL对其3D模型进行预测，并通过PyMOL软件对相关结果作了可视化处理。Autodock工具，Swiss-Pdb以及PyMOL均用以酶与邻苯二甲酸单乙基己基酯（monoethylhexyl phthalate，MEHP）的相互作用分析。[结果]该酶的初级结构与已报道Gordonia sp. P8219菌株中的酯类水解酶相似，该酶的最适温度与pH （分别为40℃和8.0）和菌株P8219有所不同；该酶在有机溶剂、洗涤剂和金属离子环境中表现出良好的稳定性；然而，其酶活受2 mol/L的Ni²⁺、Fe³⁺、Cu²⁺和Zn²⁺离子，1 mol/L苯甲基磺酰氟（PMSF），0.5 mol/L的对氧磷，1 mol/L苯基醚（PGO），2 mol/L焦碳酸二乙酯（DEPC）和5 mol/L的毒扁豆碱所抑制。丝氨酸水解酶中高度保守五肽基序GXSXG与催化三联体HSD结构均存在于该酶的编码序列中。根据分子对接结果，Thr152与Ser230在水解酶与其模板之间也显得更为保守，且与MEHP联系较为紧密（分别为5.8 Å和3.6 Å），可能起着重要的催化作用。而MEHP与催化氨基酸残基Ser125、His291、Asp259并不十分靠近。[结论]YC-RL2中MehpH在有机溶剂、洗涤剂以及金属离子环境中具有稳定的生物活性，表明其具有良好的应用潜力。酶的结构及活性中心得到了初步的分析，对于催化机理及酶改造研究具有重要意义。

Abstract:

[Objective] This study aimed to investigate the effect of environmental factors on a monoethylhexyl phthalate hydrolase (MehpH) activity, model the 3-D structure of the enzyme and the interaction of the catalytic amino acid residues with the substrate.[Methods] The effect of environmental factors was determined by the standard enzyme assay. Primary structure analysis and 3-D model prediction were completed by DNAMAN (Version 2.1) and SWISS-MODEL server respectively and the results visualized by PyMOL software. Autodock tools, Swiss-PDB viewer and PyMOL were used to investigate the interactions between the enzyme and monoethylhexyl phthalate (MEHP).[Results] The primary structure of this enzyme was similar to MehpH from Gordonia sp. P8219 with different optimum temperature and pH (40℃ and 8.0, respectively). The enzyme was stable in presence of organic solvents, detergents and ions. However, it was inhibited by 2 mol/L of Ni²⁺, Fe³⁺, Cu²⁺, Zn²⁺ ions, 1 mol/L phenylmethylsulfonyl fluoride (PMSF), 0.5 mol/L paraoxon, 1 mol/L phenyl glyoxal (PGO), 2 mol/L diethyl pyrocarbonate (DEPC) and 5 mol/L eserine. The pentapeptide motif GXSXG and catalytic triad HSD conserved in serine hydrolases were present in the sequence. The docking result showed that the amino residues Thr152 and Ser230 were much conserved among the hydrolases and closely associated with MEHP (5.8 Å and 3.6 Å respectively) and thus may play important roles in the catalytic process. However, MEHP was not very close to the catalytic triad acid residues Ser125, His291, Asp259.[Conclusion] This study showed that MehpH in YC-RL2 was fairly stable in presence of organic solvents, detergents and metal ions indicating its application potential. The structural and catalytic analysis provides important information for further investigation of catalytic mechanism and enzymatic modification.