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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways.
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Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways.

机译：工程化的DsbC嵌合体可催化大肠杆菌中的蛋白质氧化和二硫键异构化：调节两个竞争途径。

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摘要

In the Escherichia coli periplasm, the formation of protein disulfide bonds is catalyzed by DsbA and DsbC. DsbA is a monomer that is maintained in a fully oxidized state by the membrane enzyme DsbB, whereas DsbC is a dimer that is kept reduced by a second membrane protein, DsbD. Although the catalytic regions of DsbA and DsbC are composed of structurally homologous thioredoxin motif domains, DsbA serves only as an oxidase in vivo, whereas DsbC catalyzes disulfide reduction and isomerization and also exhibits significant chaperone activity. To reconcile the distinct catalytic activities of DsbC and DsbA, we constructed a series of chimeras comprising of the dimerization domain of DsbC, with or without the adjacent alpha-helical linker region, fused either to the first, second, third, or fifth residue of intact DsbA or to thioredoxin. The chimeras fully substituted for DsbC in disulfide-bond rearrangement and also were able to restore protein oxidation in a dsbA background. Remarkably, the chimeras couldserve as a single catalyst for both disulfide-bond formation and rearrangement, thus reconciling the kinetically competing DsbB-DsbA and DsbD-DsbC pathways. This property appeared to depend on the orientation of the DsbA active-site cysteines with respect to the DsbC dimerization domain. In vitro, the chimeras had high chaperone activity and significant reductase activity but only 15-22% of the disulfide-isomerization activity of DsbC, suggesting that rearrangement of nonnative disulfides may be mediated primarily by cycles of random reduction and reoxidation.
机译：在大肠杆菌周质中，DsbA和DsbC催化蛋白质二硫键的形成。 DsbA是被膜酶DsbB保持在完全氧化状态的单体，而DsbC是被第二种膜蛋白DsbD保持还原的二聚体。尽管DsbA和DsbC的催化区域由结构同源的硫氧还蛋白基序结构域组成，但DsbA仅在体内充当氧化酶，而DsbC催化二硫键还原和异构化，并且还具有显着的伴侣活性。为了调和DsbC和DsbA的独特催化活性，我们构建了一系列嵌合体，包括DsbC的二聚结构域，带有或不带有相邻的α-螺旋接头区域，融合到DsbC的第一个，第二个，第三个或第五个残基上完整的DsbA或硫氧还蛋白。嵌合体在二硫键重排中完全替代了DsbC，并且还能够在dsbA背景下恢复蛋白质氧化。引人注目的是，嵌合体可以作为二硫键形成和重排的单一催化剂，从而调和动力学上竞争的DsbB-DsbA和DsbD-DsbC途径。此属性似乎取决于DsbA活性位点半胱氨酸相对于DsbC二聚结构域的方向。在体外，嵌合体具有较高的伴侣活性和显着的还原酶活性，但仅占DsbC二硫键异构化活性的15-22％，这表明非天然二硫键的重排可能主要是由随机还原和再氧化循环介导的。

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