Bader M, Muse W, Zander T, Bardwell J
Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, USA.
J Biol Chem. 1998 Apr 24;273(17):10302-7. doi: 10.1074/jbc.273.17.10302.
Disulfide bonds are important for the structure and stability of many proteins. In prokaryotes their formation is catalyzed by the Dsb proteins. The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins. Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA. Here we demonstrate the direct reoxidation of DsbA by DsbB. We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA. We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion. The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction. Membranes from a dsbB null mutant display no DsbA reoxidation activity. The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a Km = 10 microM. The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.
二硫键对许多蛋白质的结构和稳定性都很重要。在原核生物中，它们的形成由Dsb蛋白催化。DsbA蛋白作为二硫键的直接供体，作用于新合成的周质蛋白。遗传学证据表明，另一种名为DsbB的蛋白专门用于使DsbA重新氧化。在此，我们证明了DsbB对DsbA的直接重新氧化作用。我们开发了一种荧光测定法，使我们能够直接跟踪DsbA的重新氧化过程。我们发现，含有催化量DsbB的膜能够迅速将DsbA完全重新氧化。该反应强烈依赖于氧气的存在，这意味着氧气是这种二硫键形成反应的最终电子受体。来自dsbB基因敲除突变体的膜没有显示出DsbA重新氧化活性。DsbB重新氧化DsbA的能力符合米氏行为，其中DsbA作为DsbB的高亲和力底物，Km = 10微摩尔。本文描述的体外重建是对DsbB的首次生化分析，使我们能够研究大肠杆菌中二硫键形成的主要途径。
