Four-Dimensional Protein Crystallography of Nitrile Hydratase Reaction

来自Rodococcus sp. N-771的腈水合酶是一种催化腈水合成相应酰胺的酶，它含有一个单核非血红素铁作为反应中心（fe -型nase）。中心是光反应性的，亚硝基化灭活，光驱动NO释放激活。光活化的铁型nase在有氧条件下24小时内失去活性。先前的研究表明，翻译后修饰的活性酶亚硫酸半胱氨酸（aCys114-SO-）在有氧条件下进一步氧化为亚硫酸半胱氨酸（aCys114-SO_2-）。为了避免进一步氧化，在氧浓度小于0.1% （v/v）的厌氧条件下构建了结晶体系。通过x射线晶体学研究了完整fe型nase的真正活性结构，包括以丁酸为抑制剂/稳定剂和以环己基异氰酸酯（ch-NC）为底物类似物的复合结构。我们还在厌氧条件下与ch-NC以络合物形式结晶了非活性亚硝基化NHase。利用大角度振荡技术（LOT）在RIKEN光束线：BL45XU， SPring-8上以30min的时间分辨率跟踪了光激活后的动态结构变化。在此基础上，揭示了acys114 - so -在fe型nase的腈水化机制中的作用。

Nitrile hydratase from Rodococcus sp. N-771 is the enzyme that catalyzes the hydration of nitriles to the corresponding amides, and contains a mononuclear non-heme iron as the reaction center (Fe-type NHase). The center is photo-reactive, inactivated by nitrosylation and activated by photo-driven NO release. The photo-activated Fe-type NHase loses the activity within 24 hours under aerobic conditions. Previous studies have revealed that the post-translationally modified cystein sulfenate (aCys114-SO-) of active enzyme is further oxidized under the aerobic conditions to cystein sulfinate (aCys114-SO_2-).In order to avoid the further oxidation, a crystallization system was constructed under anaerobic conditions of less than 0.1% (v/v) oxygen concentration. The really active structure of intact Fe-type NHase was studied by X-ray crystallography, including complex structures with butyric acid as an inhibitor/stabilizer and with cyclohexyl-isocyanide (ch-NC) as a substrate analogue. We also crystallized the inactive nitrosylated NHase under the anaerobic conditions in the complex form with ch-NC. The dynamic structure changes were traced by using the large-angle oscillation technique (LOT) after photo-activation at a time-resolution of 30min at a RIKEN beamline: BL45XU, SPring-8, the data collection system of which was remodeled for our purpose. Based on the results obtained, the role of aCys 114-SO- in the nitrile hydration mechanism of Fe-type NHase was revealed.