Bessette P H, Qiu J, Bardwell J C, Swartz J R, Georgiou G
Department of Chemical Engineering, University of Texas, Austin, Texas 78712, USA.
J Bacteriol. 2001 Feb;183(3):980-8. doi: 10.1128/JB.183.3.980-988.2001.
We have examined the role of the active-site CXXC central dipeptides of DsbA and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm. DsbA active-site mutants with a wide range of redox potentials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsbA promoter by integration of the respective alleles into the bacterial chromosome. The dsbA alleles gave significant differences in the yield of active murine urokinase, a protein containing 12 disulfides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed. These results suggest that the active-site CXXC motif of DsbA can play an important role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic conditions, there was no significant difference among the DsbA mutants with respect to phenotypes depending on the oxidation of proteins with few disulfide bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined. A library of DsbC expression plasmids with the active-site dipeptide randomized was screened for mutants that have increased disulfide isomerization activity. A number of DsbC mutants that showed enhanced expression of a variant of human tissue plasminogen activator as well as mouse urokinase were obtained. These DsbC mutants overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collectively, these data indicate that the active sites of the soluble thiol- disulfide oxidoreductases can be modulated to enhance disulfide isomerization and protein folding in the bacterial periplasmic space.
我们研究了二硫键异构酶A（DsbA）和二硫键异构酶C（DsbC）活性位点的CXXC中心二肽在大肠杆菌周质中二硫键形成和异构化过程中的作用。具有广泛氧化还原电位的DsbA活性位点突变体，通过多拷贝质粒上的trc启动子表达，或者通过将各自的等位基因整合到细菌染色体中，由内源性dsbA启动子表达。这些dsbA等位基因在活性鼠尿激酶（一种含有12个二硫键的蛋白质）的产量上产生了显著差异，其中一些等位基因显著提高了尿激酶的表达水平，超过了野生型DsbA所允许的水平。未观察到dsbA变体的体外氧化还原电位与尿激酶产量之间存在直接相关性。这些结果表明，DsbA的活性位点CXXC基序在决定多二硫键蛋白的折叠过程中可发挥重要作用，其方式独立于DsbA的氧化还原电位。然而，在有氧条件下，对于依赖少数二硫键的蛋白质氧化的表型，DsbA突变体之间没有显著差异。我们还研究了DsbC的CXXC基序中活性位点突变对体内二硫键异构化的影响。通过筛选具有增加的二硫键异构化活性的突变体，构建了活性位点二肽随机化的DsbC表达质粒文库。获得了一些显示人组织纤溶酶原激活剂变体以及小鼠尿激酶表达增强的DsbC突变体。这些DsbC突变体绝大多数在二肽的C末端位置含有一个芳香族残基，而N末端残基则更多样化。总体而言，这些数据表明，可溶性硫醇 - 二硫键氧化还原酶的活性位点可以被调节，以增强细菌周质空间中的二硫键异构化和蛋白质折叠。
