Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
Mol Microbiol. 2010 Jan;75(1):13-28. doi: 10.1111/j.1365-2958.2009.06952.x. Epub 2009 Dec 3.
Current dogma dictates that bacterial proteins with misoxidized disulfide bonds are shuffled into correctly oxidized states by DsbC. There are two proposed mechanisms for DsbC activity. The first involves a DsbC-only model of substrate disulfide rearrangement. The second invokes cycles of reduction and oxidation of substrate disulfide bonds by DsbC and DsbA respectively. Here, we addressed whether the second mechanism is important in vivo by identifying whether a periplasmic reductase could complement DsbC. We screened for naturally occurring periplasmic reductases in Bacteroides fragilis, a bacterium chosen because we predicted it encodes reductases and has a reducing periplasm. We found that the B. fragilis periplasmic protein TrxP has a thioredoxin fold with an extended N-terminal region; that it is a very active reductase but a poor isomerase; and that it fully complements dsbC. These results provide direct in vivo evidence that correctly folded protein is achievable via cycles of oxidation and reduction.
目前的教条认为，具有错误氧化二硫键的细菌蛋白被 DsbC 重新排列成正确氧化的状态。DsbC 的活性有两种提出的机制。第一种涉及 DsbC 仅作为底物二硫键重排的模型。第二种则分别涉及 DsbC 和 DsbA 对底物二硫键的还原和氧化循环。在这里，我们通过鉴定周质还原酶是否可以补充 DsbC，来确定第二种机制在体内是否重要。我们在脆弱拟杆菌中筛选天然存在的周质还原酶，选择脆弱拟杆菌是因为我们预测它编码还原酶且具有还原周质。我们发现，脆弱拟杆菌周质蛋白 TrxP 具有带有扩展 N 端区域的硫氧还蛋白折叠；它是一种非常活跃的还原酶，但异构酶活性差；并且它可以完全补充 dsbC。这些结果提供了直接的体内证据，证明通过氧化和还原循环可以实现正确折叠的蛋白质。
