Regulation mechanism of the DsbA/DsbB system that catalyzes disulfine bond formation

DsbA/DsbB系统催化二硫键形成的调控机制

• 批准号: 12480188
• 负责人: ITO Koreaki
• 金额: $ 5.31万
• 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: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12480188/
• 关键词: disulfide bond; Respiratory chain; Redox regulation; Periplasm; E.coli; protein folding; タンパク質フォールディング; キノン; 膜タンパク質; DsbA; DsbB

In the Escherichia coli protein disulphide bond formation pathway, a membrane protein DsbB reoxidizes reducd periplasmic DsbA, the disulphide boud-introducing enzyme. Our results indicate that the oxidizing equivalent for disulphide bond formation is provided by oxygen through the respiratory electron transfer system in aerobically growin E. coli cells. The Cys-41-Val-Leu-Cys-44 motif in DsbB is strongly oxidized by respiratory quinone molecules. The results of our insertion mutagenesis indicated that a segment just C-terminally adjacent to CXXC motif is crucial for the respiration-coupled oxidation of DsbB(4)Ala substitution for one or all of the I le45-tyr46-Glu47-Arg48 residues, which are franked by the CXXC motif and the presumed membrane-panning region, did not abolish the respiratory coupling. In contrast,deletion of one or more residues from this segment as well as insertions of one or more Ala into it severely impaired the oxidation.Thus,the latter mutant proteins accumulated as reduced forms or last the characteristic dithiothreitol resistance. We prose that the importance of the Ile45-tyr46-Glu47-Arg48 segment may lie in its physical length rather than the chemical nature of each amino acid. It may be conceivable that a proper positioning of the CXXC motif relative to the membrane surface is crucial for its efficient interaction with membrane-anchored quinonc molecules.The reaction Could then be driven by the large difference between the redox potentials of ubiquinone ubiquinol vs(CXXC)ox/(CXXC)red.

在大肠杆菌蛋白二硫键的形成途径中，膜蛋白DsbB重新氧化还原的周质DsbA，DsbA是二硫键的引入酶。我们的结果表明，在需氧生长的大肠杆菌细胞中，形成二硫键的氧化当量是由氧气通过呼吸电子转移系统提供的。DsbB中的Cys-41-Val-Leu-Cys-44基序被呼吸醌分子强烈氧化。我们的插入突变结果表明，恰好与CXXC基序C末端相邻的一个片段对于DsbB(4)的呼吸偶联氧化至关重要，Ala替代了一个或所有由CXXC基序和推定的膜平移区域标记的Ile45-tyr46-Glu47-Arg48残基，并没有取消呼吸偶联。相反，该片段中一个或多个残基的缺失以及一个或多个ALA的插入严重削弱了氧化作用。因此，后者的突变蛋白以还原形式积累，或最终表现为对二硫苏糖醇的抗性。我们认为Ile45-tyr46-Glu47-Arg48片段的重要性可能在于它的物理长度，而不是每个氨基酸的化学性质。可以想象的是，CXXC基序相对于膜表面的适当位置对于它与膜锚定的醌分子的有效相互作用是至关重要的，然后反应可能是由泛醌泛喹酚和(CXXC)ox/(CXXC)red之间的氧化还原电位的巨大差异所驱动的。

项目成果

Histoshi Nakatogawa: "Secretion monitor, SecM, undergoes self translation arrest in the cytosol"Mol. Cell. 7. 185-192 (2001)

Histoshi Nakatokawa：“分泌监视器，SecM，在细胞质中经历自翻译停滞”Mol。

Gen Matsumoto: "Mutation in secY that causes enhanced SecA insertion and impaired late functions in protein translocation"Journal of Bacteriology. 182. 3377-3382 (2000)

Gen Matsumoto：“secY 突变导致 SecA 插入增强并损害蛋白质易位的后期功能”《细菌学杂志》。

Taeko Kobayashi: "Identification of a segment of DsbB essential for its respiration-coupled oxidation"Molecular Microbiology. 39. 158-165 (2001)

Taeko Kobayashi：“鉴定其呼吸耦合氧化所必需的 DsbB 片段”分子微生物学。

Hiroyuki Mori: "An essential amino acid residue in protein translocation channel revealed by targeted random mutagenesis of SecY"Proc. Natl. Acad. Sci. USA. 98. 5128-5133 (2001)

Hiroyuki Mori：“SecY 的定向随机诱变揭示了蛋白质易位通道中的必需氨基酸残基”Proc。

Hitoshi Nakatogawa: "Secretion monitor, SecM, undergoes self translation arrest in the cytosol"Molecular Cell. 7. 185-192 (2001)

Hitoshi Nakatokawa：“分泌监视器，SecM，在细胞质中经历自翻译停滞”分子细胞。