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标题：Engineered DsbC chimeras catalyze both protein oxidation and disulfide-bond isomerization in Escherichia coli: Reconciling two competing pathways
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作者：Laura Segatori ; Paul J. Paukstelis ; Hiram F. Gilbert 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：27
页码：10018-10023
DOI：10.1073/pnas.0403003101
语种：English
出版社：The National Academy of Sciences of the United States of America
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 could serve 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 rear-rangement of nonnative disulfides may be mediated primarily by cycles of random reduction and reoxidation.