Mechanism of iron-sulfur cluster biosynthesis : Analysis of a framework and the reaction mechanism for the two iron-sulfur cluster assembly systems.

铁硫簇生物合成机制：分析两种铁硫簇组装系统的框架和反应机制。

• 批准号: 15510174
• 负责人: TAKAHASHI Yasuhiro
• 金额: $ 2.18万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510174/
• 关键词: iron-sulfur cluster; iron-sulfur protein; biosynthesis; ISC machinery; SUF machinery; structure-function; X-ray crystal structure; 分子間相互作用

Iron-sulfur (Fe-S) proteins are present in almost all living organisms and exhibit diverse functions, which include electron transport, redox and non-redox catalysis, and sensing for regulatory processes. They contain clusters of iron and sulfur atoms with variable complexity, such as [2Fe-2S], [3Fe-4S], and [4Fe-3S] clusters. We have recently found two independent systems, called ISC and SUF, essential for the biosynthesis of Fe-S clusters, although still very little is known about the mechanistic details. In this study we have undertaken genetic, biochemical and structural approaches to elucidate the complex mechanism by which the Fe-S clusters are assembled.1. We have isolated and analyzed 14 psuedorevertants generated from a mutant strain lacking HscA and HscB in the ISC machinery, which revealed various suppressor mutations located in IscU. We also found that the IscU homolog from the thermophilic bacterium Aquifex aeolicus exceptionally carries the intermediate Fe-S cluster in the stable form that was further stabilized by introduction of several mutations.2. Purification and biochemical characterization of the components of the SUF machinery have revealed dimeric form of SufD and the SufC_2SufD_2 oligomer as well as the SufB_1SufC_2 SufD_1 complex, suggesting that dynamic alteration of the oligomeric status is involved in the reaction. Essential residues for the SufD function have also been identified by mutagenic studies.3. We have determined the crystal structures of E.coli yfhJ (IscX), SufA, SufC, and SufD. The structure of SufA revealed the active-site architecture comprising of four cysteine residues. We have also identified a salt bridge that appears to trigger the conformational change of SufC. The structure of SufD revealed an unprecedented β-helix dimer. The docking model between SufC and SufD suggests the role of SufC to alter the oligomeric status of SufD.

铁硫（FE-S）蛋白几乎在所有活生物体和暴露的潜水功能中都呈现，包括电子传输，氧化还原和非雷克斯催化剂，以及用于调节过程的感知。它们包含具有可变复杂性的铁原子和硫原子簇，例如[2FE-2S]，[3FE-4S]和[4FE-3S]簇。我们最近发现了两个独立的系统，称为ISC和SUF，对于Fe-S簇的生物合成至关重要，尽管对机械细节知之甚少。在这项研究中，我们采用了遗传，生化和结构方法，以阐明Fe-S簇组装的复杂机制。1。我们已经分离并分析了14个由缺乏HSCA和HSCB的突变菌株产生的伪变量，ISC机械中揭示了位于ISCU中的各种抑制突变。我们还发现，来自嗜热细菌的ISCU同源物异常以稳定形式携带中间的Fe-S簇，通过引入多个突变进一步稳定。2。 Suf机械组件的纯化和生化表征已显示出SUFD的二聚体形式和SUFC_2SUFD_2低聚物以及SUFB_1SUFC_2 SUFD_1复合物，这表明寡头状态的动态变化与反应有关。 SUFD功能的基本残差也通过诱变研究确定。3。我们已经确定了大肠杆菌YFHJ（ISCX），SUFA，SUFC和SUFD的晶体结构。 Sufa的结构揭示了四个半胱氨酸保留的活跃部位结构。我们还确定了一座似乎触发SUFC会议变化的盐桥。 SUFD的结构显示出了前所未有的β-螺旋二聚体。 SUFC和SUFD之间的对接模型表明，SUFC在改变SUFD的寡聚状态的作用。

项目成果

Vahab Ali: "An Intestinal Parasitic Protist Entamoeba histolytica Possesses a Non-redundant NIF-like System for Iron-Sulfur Cluster Assembly under Anaerobic Conditions"J.Biol.Chem.. (in press). (2004)

Vahab Ali：“肠道寄生原生生物溶组织内阿米巴拥有非冗余 NIF 类系统，可在厌氧条件下组装铁硫簇”J.Biol.Chem..（出版中）。

Takahiro Yano: "Characterization of cluster N5 as a fast-relaxing [4Fe4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans"J.Biol.Chem.. 278・18. 15514-15522 (2003)

Takahiro Yano：“脱氮副球菌质子转位 NADH-泛醌氧化还原酶 Nqo3 亚基中 N5 簇的表征”J.Biol.Chem.. 15514-15522（2003 年） ）

Marco Kriek: "Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase"Protein Expr.Purif.. 28・2. 241-245 (2003)

Marco Kriek：“铁硫簇组装蛋白对大肠杆菌硫辛酸合酶表达的影响”Protein Expr.Purif.. 28・245 (2003)。

Effect of iron-sulfur cluster assembly proteins on the expression of Escherichia coli lipoic acid synthase

• DOI: 10.1016/s1046-5928(02)00680-0
• 发表时间: 2003-04-01
• 期刊: PROTEIN EXPRESSION AND PURIFICATION
• 影响因子: 1.6
• 作者: Kriek, M;Peters, L;Roach, PL
• 通讯作者: Roach, PL

An Intestinal Parasitic Protist Entamoeba histolytica Possesses a Non-redundant NIF-like System for Iron-Sulfur Ouster Assembly under Anaerobic Conditions

肠道寄生原生生物溶组织内阿米巴拥有非冗余的类 NIF 系统，可在厌氧条件下进行铁硫驱逐组装

• 发表时间: 2004
• 期刊: J.Biol.Chem. 279
• 作者: Yoshimitsu Shimomura;Yoshimitsu Shimomura;Yoshimitsu Shimomura;Vahab Ali
• 通讯作者: Vahab Ali