Dynamism of activated-selenium species: Structural biological analysis of mechanism of biosynthesis of selenium-containing proteins

活化硒物种的动态：含硒蛋白质生物合成机制的结构生物学分析

• 批准号: 13480192
• 负责人: ESAKI Nobuyoshi
• 金额: $ 10.94万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13480192/
• 关键词: selenocysteine; selenocysteine lyase; selenium; selenoprotein; activated-selenium species; 部位特異的変異; X線結晶構造解析; プロパルギルグリシン

Selenocysteine lyase (SCL) is an enzyme which specifically catalyzes the decomposition of selenocysteine to form alanine and selenium. The enzyme is proposed to be involved in an initial step of the biosynthesis of selenoproteins containing selenocysteine residues because it has an activity to deliver selenide for selenophosphate synthetase. Escherichia coli contains its homologous counterpart enzyme, cysteine desulfurase (CSD). Unlike SCL, CSD acts on both cysteine and selenocysteine as substrates. A conserved cysteine residue important for the decomposition of cysteine has been demonstrated to be not essential for the decomposition of selenocyteine. Mouse SCL has eight cysteine residues. We constructed mutant enzymes C101S, C129S, C177S, C304S, C346, C364S, C375, and C390S by site-directed mutagenesis. The mutant enzymes were purifued and characterized. The C375S and C375A mutants lost the selenocysteine lyase activity. Therefore, Cys375 is essential for the selenocysteine lyase activity of the enzyme, indicating that the mechanism of selenocysteine lyase reaction by SCL is different from that catalyzed by CSD.

硒半胱氨酸裂解酶(SCL)是一种特异性催化硒半胱氨酸分解为丙氨酸和硒的酶。该酶被认为参与了含硒半胱氨酸残基的硒蛋白生物合成的初始步骤，因为它具有为硒磷酸合成酶输送硒的活性。大肠杆菌含有其同源酶半胱氨酸脱硫酶(CSD)。与SCL不同，CSD同时作用于半胱氨酸和硒半胱氨酸作为底物。一个保守的半胱氨酸残基对半胱氨酸的分解很重要，已被证明对硒半胱氨酸的分解不是必需的。小鼠SCL有8个半胱氨酸残基。我们通过定点突变构建了突变酶C101S、C129S、C177S、C304S、C346、C364S、C375和C390S。对突变的酶进行了纯化和鉴定。C375S和C375A突变株失去了硒半胱氨酸裂解酶的活性。因此，Cys375对该酶的硒半胱氨酸裂解酶活力是必需的，表明SCL与CSD催化硒半胱氨酸裂解酶的反应机理不同。

项目成果

H.Mihara et al.: "Role of cysteine desulfurase in the biosynthesis of formate dehydrogenase H containing Mo, molybdopterin, selenocysteine, and Fe_4S_4 cluster"Biomed. Res. Trace Elements. 12・4. 261-262 (2001)

H. Mihara等人：“半胱氨酸脱硫酶在含有Mo、钼蝶呤、硒代半胱氨酸和Fe_4S_4簇的生物合成中的作用”Biomed Trace Elements 12・4。

E.A.Pion-Smits et al.: "Characterization of a NifS-like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism"Plant Physiol.. 130・3. 1309-1318 (2003)

E.A.Pion-Smits 等：“来自拟南芥的 NifS 样叶绿体蛋白的表征。其在硫和硒代谢中的作用的意义”植物生理学.. 130・3（2003）。

Masami Kashiwa et al.: "Removal of soluble selenium by a selenate-reducing bacterium Bacillus Sp. SF-1"Biofactors. 14・1-4. 261-265 (2001)

Masami Kashiwa 等：“硒酸还原菌 Bacillus Sp. SF-1 去除可溶性硒”Biofactors 14・1-4 (2001)。

Hisaaki Mihara et al.: "Structure of External Aldimine of Escherichia coli CsdB, an IscS/NifS Homolog: Implications for Its Specificity Toward Selenocysteine"Journal of Biochemistry. (印刷中). (2002)

Hisaaki Mihara 等人：“大肠杆菌 CsdB 的外部醛亚胺结构，IscS/NifS 同源物：其对硒代半胱氨酸的特异性的影响”《生物化学杂志》（2002 年出版）。

H.Mihara et al.: "The iscS gene is essential for the biosynthesis of 2-selenouridine in tRNA and the selenocysteine-containing formate dehydrogenase H"Proc. Natl. Acad. Sci. U.S.A.. 99・10. 6679-6683 (2002)

H. Mihara 等：“iscS 基因对于 tRNA 中的 2-硒代尿苷和含硒代半胱氨酸的甲酸脱氢酶 H 的生物合成至关重要”，Proc. Natl. 99·10。 ）