Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site
Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site
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DOI：
10.1093/emboj/cdg275
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发表时间：
2003-06-02
期刊：
影响因子：
11.4
通讯作者：
Mowbray, SL
中科院分区：
文献类型：
--
作者：
Arand, M;Hallberg, BM;Mowbray, SL
环氧化物水解酶对于解毒或代谢中含环氧化物化合物的加工至关重要。经典的环氧化物水解酶具有 α/β 水解酶折叠，并通过包括酶-底物中间体的两步反应机制发挥作用。我们在此报告了来自红平红球菌的柠檬烯-1,2-环氧化物水解酶的结构，该结构使用硒代蛋氨酸取代的蛋白质的单波长异常色散进行解析，并以 1.2 埃分辨率进行精制。这种酶代表了一种完全不同的结构和一种新颖的一步机制。该折叠具有高度弯曲的六链混合β片层，其上挤满了四个α螺旋以形成深袋。尽管这个口袋内衬的大多数残基都是疏水性的，但包括天冬氨酸-精氨酸-天冬氨酸三联体在内的一组极性基团在其最深点相互作用。定点诱变支持了这是活性位点的结论。此外，1.7埃分辨率结构显示抑制剂丙戊酸结合在该位置，其极性原子直接与三联体的残基相互作用。我们认为目前未知功能的几种细菌蛋白将具有这种结构，并且在某些情况下具有催化特性。
Epoxide hydrolases are essential for the processing of epoxide-containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an alpha/beta hydrolase fold and act via a two-step reaction mechanism including an enzyme-substrate intermediate. We report here the structure of the limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis, solved using single-wavelength anomalous dispersion from a selenomethionine-substituted protein and refined at 1.2 Angstrom resolution. This enzyme represents a completely different structure and a novel one-step mechanism. The fold features a highly curved six-stranded mixed beta-sheet, with four alpha-helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp-Arg-Asp triad, interact at its deepest point. Site-directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Angstrom resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.