Four-Dimensional Protein Crystallography of Nitrile Hydratase Reaction

腈水合酶反应的四维蛋白质晶体学

• 批准号: 14380321
• 负责人: KAMIYA Nobuo
• 金额: $ 8.51万
• 依托单位: RIKEN
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380321/
• 关键词: Nitrile Hydratase; Photo-activation; Four-dimensional Crystallography; SPring-8; Large-angle Oscillation Technique; Site-directed mutant protein; SPning-8; 小型カッパーゴニオメータ; イソニトリルヒドラターゼ

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.

产腈水合酶的红球菌。N-771是一种催化氰化物水合生成相应酰胺的酶，它含有一个单核非血红素铁作为反应中心(Fe型NHase)。该中心是光反应的，通过亚硝化而失活，通过光驱动的NO释放而激活。在好氧条件下，光活化的Fe型NHase在24小时内失去活性。以往的研究表明，活性酶的翻译后修饰的半胱氨酸硫酸盐(aCys114-SO-)在有氧条件下进一步氧化为半胱氨酸亚硫酸盐(aCys114-SO2-)。为了避免进一步的氧化，在氧气浓度低于0.1%(v/v)的厌氧条件下构建了结晶系统。用X射线结晶学研究了完整的Fe型NHase的活性结构，包括以丁酸为抑制剂/稳定剂和以环己基异氰酸酯(ch-NC)为底物类似物的络合物结构。我们还将厌氧条件下失活的亚硝酸氨酶与ch-NC形成络合物。利用大角度振荡技术(LOT)，在RIKEN BL45XU SPRING-8光束线上，以30min的时间分辨率对光激活后的动态结构变化进行了跟踪，其数据采集系统是为我们的目的而改装的。在此基础上，揭示了Acys 114-SO-在铁型氨酶的丁腈水合机理中的作用。

项目成果

Crystal structure of Thermus themophilus HB8 H-protein of the glycine cleavage system, resolved by a six-dimensional molecular-replacement method

嗜热栖热菌 HB8 H 蛋白甘氨酸裂解系统的晶体结构，通过六维分子置换法解析

• 发表时间: 2003
• 期刊: Acta Cryst.Sec.D 59
• 作者: Nakai;T.;Ishijima;J.;Masui;R.;Kuramitsu;S.;Kamiya;N.
• 通讯作者: N.

Oinuma, K-I., Hashimoto, Y., Konishi, K., Goda, M., Noguchi, T., Higashibata, H., Kobayashi, M.: "Novel aldoxime dehydratase involved in carbon-nitrogen triple bond synthesis of Pseudomonas chlororaphis B23 : Sequencing, gene expression, purification and

Oinuma, K-I.、Hashimoto, Y.、Konishi, K.、Goda, M.、Noguchi, T.、Higashibata, H.、Kobayashi, M.：“参与绿针假单胞菌碳氮三键合成的新型醛肟脱水酶

Crystal structure of Thermus thermophilus HB8 H-protein of the glycine cleavage system, resolved by a six-dimensional molecular-replacement method

嗜热栖热菌 HB8 H 蛋白甘氨酸裂解系统的晶体结构，通过六维分子置换法解析

• 发表时间: 2003
• 期刊: Acta Cryst. Sec. D 50
• 作者: Nakai;T.;Ishijima;J.;Masui;R.;Kuramitsu;S.;Kamiya;N.
• 通讯作者: N.

Nakai, T., Ishijima, J., Masui, R., Kuramitsu, S., Kamiya, N.: "Crystal structure of Thermus thermophilus HB8 H-protein of the glycine cleavage system, resolved by a six-dimensional molecular replacement method"Acta Cryst.Sec.D. 59. 1610-1618 (2003)

Nakai, T.、Ishijima, J.、Masui, R.、Kuramitsu, S.、Kamiya, N.：“甘氨酸裂解系统的嗜热栖热菌 HB8 H 蛋白的晶体结构，通过六维分子置换方法解析

Goda, M., Hashimoto, Y., Shimizu, S., et al.: "Isonitrile hydratase from Pseudomonas putida N19-2: Cloning, sequencing, gene expression, and identification of its active amino acid residue"The Journal of Biological Chemistry. 276・(26). 45860-45865 (2002)

Goda, M.、Hashimoto, Y.、Shimizu, S. 等人：“来自恶臭假单胞菌 N19-2 的异腈水合酶：其活性氨基酸残基的克隆、测序、基因表达和鉴定”生物化学杂志276·(26)。