Studies on the structure and function relationship offumarate redudase from adut Ascais suum

猪蛔虫成虫富马酸还原酶结构与功能关系的研究

• 批准号: 18370042
• 负责人: HARADA Shigeharu
• 金额: $ 4.22万
• 依托单位: Kyoto Institute of Technology
• 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-18370042/
• 关键词: fiat-mate rpduntacp; elpntrnn transfpr system; X-ray structure analysis; X線結晶構造解析; 寄生虫蛋白質; キノール酸化酵素

In eukaryotes, complex II is localized in the inner mitochondrial membrane, and is generally composed of 4 subunits (two hydrophilic subunits, Fp and lp, and two hydrophobic membrane anchoring subunits, CybL and CybS). The enzyme catalyzes the oxidation of succinate to fumarate in conjunction with the reduction of ubiquinone to ubiquinol during aerobic respiration. On the other hand, Complex II from adult Ascaris suum, living in anaerobic small intestine, catalyzes the reverse reaction, the reduction of fumarate to succinate in conjunction with the oxidation of rhodoquinol to rhodoquinone. In this study, Complex II was purified, crystallized and its three-dimensional structure was determined. Crystals suitable for X-ray structure analysis were obtained by the dialysis method using PEG3350 as a precipitant in the presence of octaethyleneglycol monododecyl ether and dodecy maltoside. X-ray diffraction data were collected to a resolution of 2.8 A at beamline BL44XU (Spring-8, Japan). The structure was solved by the molecular replacement method using the refined coordinates of the porcine Complex II. After refinement, the final structure gave R-factor of 25.0%(R-free=28.8%). The structure of the adult A. suum Complex II is essentially identical to those determined to data such as from Escherichia coli, porcine and avian etc. Especially, cofactors (one FAD and three iron-sulfur clusters) located between rhodoquinol and fumarate binding sites are surrounded by amino acid residues well conserved in Complex Iis from many species. Further, fumarate bound to the Fp subunit faces to the FAD isoalloxazine ring and takes twisted conformation. The rhodoquinone binding site formed by the Ip, CybL and CybS subunits is also mainly constructed by well conserved amino acid residues. Based on the three-dimensional structure of Complex II from A. suum mitochondria, structural insight into the mechanism of the oxidation of rhodoquinol to rhodoquinone could be obtained.

在真核生物中，复合物II位于线粒体内膜中，并且通常由4个亚基（两个亲水性亚基Fp和lp，以及两个疏水性膜锚定亚基CybL和CybS）组成。在有氧呼吸过程中，该酶催化琥珀酸氧化为富马酸，同时将泛醌还原为泛醇。另一方面，来自生活在厌氧小肠中的猪蛔虫成虫的复合物II催化逆反应，将反丁烯二酸还原成琥珀酸，同时将对苯二酚氧化成对苯二酚。在这项研究中，复合物II的纯化，结晶和其三维结构进行了测定。在八甘醇单十二烷基醚和十二烷基麦芽糖苷的存在下，使用PEG 3350作为沉淀剂，通过透析法获得适合于X射线结构分析的晶体。在光束线BL 44 XU（Spring-8，Japan）处收集X射线衍射数据，分辨率为2.8 A。使用猪复合物II的精确坐标通过分子置换法求解结构。精制后，最终结构的R因子为25.0%（无R =28.8%）。本文报道了A.猪复合物II与大肠杆菌、猪和禽类等的数据基本相同。特别是，位于对苯二酚和富马酸盐结合位点之间的辅因子（一个FAD和三个铁硫簇）被许多物种的复合物II中保守的氨基酸残基包围。此外，与Fp亚基结合的富马酸酯面向FAD异咯嗪环并采取扭曲构象。由Ip、CybL和CybS亚基形成的玫瑰醌结合位点也主要由高度保守的氨基酸残基构成。基于A. suum线粒体，结构洞察的机制，氧化的玫瑰醌的玫瑰醌。

项目成果

Inhibition assay of b-hematin formation initiated by lecithin for screening new antimalarial drugs

卵磷脂引发的β-血红素形成抑制试验用于筛选新的抗疟药物

• 发表时间: 2006
• 期刊: Analytical Biochemistry 349
• 作者: Dai Thi Xuan Trang;Nguyen Tien Huy;Dinh Thanh Uyen;Motohiro Sasai;Takeshi Shiono;Shigeharu Harada;Kaeko Kamei
• 通讯作者: Kaeko Kamei

回虫(Ascaris suum)成虫ミトコンドリア複合体IIの結晶構造解析

猪蛔虫成虫线粒体复合物II的晶体结构分析

• 发表时间: 2007
• 作者: Nguyen Tien Huy;Dinh Thanh Uyen;Motohiro Sasai;Dai Thi Xuan Trang;Takeshi Shiono;Shigeharu Harada;kaeko Kamei;Y. Kanaho;清水洋成
• 通讯作者: 清水洋成

Serine proteinase inhibitor from wax gourd (Benincasa hisida Thunb Cogn.) seeds

冬瓜 (Benincasa hisida Thunb Cogn.) 种子中的丝氨酸蛋白酶抑制剂

• 发表时间: 2006
• 期刊: Biosci. Biotechnol. Biochem. 70
• 作者: Panida Atiwetin;Shigeharu Harada;Kaeko Kamei
• 通讯作者: Kaeko Kamei

Alchohols induce beta-hematin formation via the dissociation of aggregated heme and reduction in interfacial tension of the solution

酒精通过聚集血红素的解离和溶液界面张力的降低诱导 β-血红素形成

• 发表时间: 2007
• 期刊: Actq Tropics 101
• 作者: Su W;et al.;Nguyen Tien Huy;Nguyen Tien Huy
• 通讯作者: Nguyen Tien Huy

Simple colorimetric inhibition assay of heme crystallization for high-throughput screening of antimalarial compounds

• DOI: 10.1128/aac.00985-06
• 发表时间: 2007-01-01
• 期刊: ANTIMICROBIAL AGENTS AND CHEMOTHERAPY
• 影响因子: 4.9
• 作者: Huy, Nguyen Tien;Uyen, Dinh Thanh;Kamei, Kaeko
• 通讯作者: Kamei, Kaeko