Four-dimensional structure analysis of Ndx family enzyme utilizing a new pH-temperature jump trigger

利用新的 pH-温度跳跃触发器对 Ndx 家族酶进行四维结构分析

• 批准号: 18370047
• 负责人: KAMIYA Nobuo
• 金额: $ 10.11万
• 依托单位: Osaka City University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18370047/
• 关键词: 4-D structure analysis; pH jump; temperature jump; Ndx family; functional genomics

Before starting our research, we tried to improve crystal quality of ADP-ribose pyro-phosphatase (ADPRase) involved in Ndx family, because we needed high resolution analysis to resolve substrates and products which would co-exist in the reaction cavity of ADPRase in time course of enzyme reaction. Many crystallization conditions were tested, and the observable diffraction limit was expanded to 1.1A resolution from 1.7A, previously reported. In order to start the crystalline state reaction of ADPRase, Zn(II) ions were soaked into the crystals, in which the substrate ADPR was introduced in advance. The reaction was stopped by a temperature jump from 295K to 90K. Crystal structures were determined at 9 points of reaction time, 0, 3, 6, 10, 15, 20, 30, 40, 60min, and the in situ observation of ADPRase reaction was succeeded. Our results showed that the enzyme reaction progressed in crystal after the Zn(II) ion soaking as following. (1) The first Zn(II) ion introduced into reaction cavity changed ADPR conformation to an intermediate state, designated as ADPR^*.(2) The second Zn(II) ion ligated ADPR^*, gultamate side chain of E82 and E86, and two water molecules making a five-ligands coordination structure. (3) One of two water molecules was activated by the second Zn(II) ion and E82 to hydroxide anion, and (4) the anion attacked nucleophilically the alpha phosphate of ADPR to brake the pyrophosphate bond to produce two products, AMP and ribose-5'-phosphate. The biochemical insight into the in situ observation of ADPRase was discussed.

在开始我们的研究之前，我们试图改善Ndx家族中的ADP-核糖焦磷酸酶（ADPRase）的晶体质量，因为我们需要高分辨率的分析来分辨在酶反应的时间过程中会共存于ADPRase反应腔中的底物和产物。许多结晶条件进行了测试，和可观察到的衍射极限从1.7A，以前报道的分辨率扩展到1.1A。为了启动ADPRase的结晶态反应，将Zn（II）离子浸入预先引入底物ADPR的晶体中。通过从295 K到90 K的温度跳变来停止反应。在反应时间0、3、6、10、15、20、30、40、60 min 9个点测定晶体结构，并成功地对ADPRase反应进行了原位观察。结果表明，Zn（Ⅱ）离子浸泡后，酶反应在晶体中进行如下。(1)引入反应腔的第一个Zn（II）离子将ADPR构象改变为中间态，命名为ADPR^*。(2)第二个Zn（II）离子与ADPR^*、E82和E86的谷氨酸侧链以及两个水分子形成五配体配位结构。(3)两个水分子中的一个被第二个Zn（II）离子和E82活化为氢氧根阴离子，并且（4）阴离子亲核攻击ADPR的α磷酸，以破坏焦磷酸键，产生两种产物，AMP和核糖-5 '-磷酸。讨论了ADPRase原位观察的生物化学观点。

项目成果

Mutational study on aGln90 of Fe-type nitrile hydratase from Rgidiciccys sp.N771

Rgidiciccys sp.N771铁型腈水合酶aGln90突变研究

• 发表时间: 2006
• 期刊: Biosci.Biotech.and Biochem. 70
• 作者: Takarada;H.;Kawano;Y.;Hashimoto;K.;Nakayama;H.;Ueda;S.;Yohda;M.;Kamiya;N.;Dohnae;N.;Maeda;M.;Odaka;M.
• 通讯作者: M.

Reaction pathway of ADP-ribose pyrophosphatase, revealed by time-resolved X-ray crystallography

时间分辨X射线晶体学揭示ADP-核糖焦磷酸酶的反应途径

• 发表时间: 2008
• 作者: Kamiya,N.;Kai,K.;Nakagawa,N.;Kuramitsu,S.;Miyahara,I
• 通讯作者: Miyahara,I

Structural Basis for Different Substrate Specificities of Two A DP-Ribose Pyrophosphatases from Thermus thermophilus HB8

嗜热栖热菌 HB8 中两种 A DP-核糖焦磷酸酶不同底物特异性的结构基础

• 发表时间: 2008
• 期刊: J.Bacteriol. 190
• 作者: Wakamatsu,T.;Nakagawa,N.;Kuramitsu,S.;Masui,R.
• 通讯作者: Masui,R.

Location of PsbY in oxygen-evolving photosystem II revealed by mutagenesis and X-ray crystallography

• DOI: 10.1016/j.febslet.2007.09.036
• 发表时间: 2007-10-16
• 期刊: FEBS LETTERS
• 影响因子: 3.5
• 作者: Kawakami, Keisuke;Iwai, Masako;Shen, Jian-Ren
• 通讯作者: Shen, Jian-Ren

クライオトラップ法により追跡したMPリボースピロリン酸分解酵素の結晶相反応経路

低温冷阱法追踪MP核糖螺磷酸降解酶的晶相反应路径

• 发表时间: 2007
• 作者: Kamiya,N.;Kai,K.;Nakagawa,N.;Kuramitsu,S.;Miyahara,I;神谷 信夫
• 通讯作者: 神谷 信夫