Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA.
Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA
J Bacteriol. 2018 Jul 25;200(16). doi: 10.1128/JB.00137-18. Print 2018 Aug 15.
Disulfide bonds influence the stability and activity of many proteins. In , the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the , use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a -like gene. and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The gene complements an deletion strain, restoring the oxidation of DsbA. While we have suggested that the gene linked to the gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the genome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways in and in , a close relative of Our results show that only DsbA is oxidized by VKOR. Comparison of the properties of - and -null mutants in shows parallels to the properties of mutations in Disulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the genome as possible substrates of the VKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway in and showed that of the five candidates, only DsbA is efficiently oxidized by VKOR in We also present evidence for the involvement of VKOR in DsbA oxidation in .
二硫键影响许多蛋白质的稳定性和活性。在大肠杆菌中，DsbA 和 DsbB 酶促进二硫键的形成。其他细菌，包括分枝杆菌，使用维生素 K 环氧化物还原酶 (VKOR) 代替 DsbB，其基因要么与 DsbA 样基因融合，要么位于同一操纵子中。肺炎克雷伯氏菌和其他革兰氏阳性放线菌将许多具有偶数半胱氨酸的蛋白质分泌到细胞包膜中。这些生物体具有预测的氧化还原酶和 VKOR 同源物。这些发现表明，这些细菌可能在细胞包膜中形成二硫键。DsbA 基因补充了一个缺失菌株，恢复了 DsbA 的氧化。虽然我们已经表明与 DsbA 基因相连的基因可能表达分枝杆菌中 VKOR 的伴侣，但其他人认为两个其他细胞外氧化还原酶 (DsbE 或 DsbF) 可能是蛋白质二硫键形成的催化剂。然而，没有直接证据表明 VKOR 与 DsbA、DsbE 或 DsbF 相互作用。为了确定 VKOR 的实际底物，我们使用基因组的生物信息学分析鉴定了另外两个预测的细胞外 DsbA 样蛋白。使用五个潜在的 DsbA，我们试图在大肠杆菌和与其密切相关的分枝杆菌中重建二硫键途径。我们的结果表明，只有 DsbA 被 VKOR 氧化。比较分枝杆菌和大肠杆菌中的 -和 -null 突变体的特性表明与分枝杆菌中的 DsbA 突变的特性相似。二硫键的形成对细菌的致病性有很大的影响。因此，形成二硫键的蛋白质代表了开发抗菌药物的新靶标，因为抑制二硫键的形成将导致几类毒力因子的同时丧失活性。在这里，我们确定了分枝杆菌基因组编码的五个候选蛋白作为参与二硫键形成的 VKOR 蛋白的可能底物。然后，我们在大肠杆菌中重建了分枝杆菌的二硫键形成途径，并表明在这五个候选蛋白中，只有 DsbA 被 VKOR 有效地氧化。我们还提供了 VKOR 参与 DsbA 氧化的证据。
