Reconstitution of a Protein Disulfide Catalytic System

Abstract

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 K m ‫؍‬ 10 M. 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.Proteins start life as linear amino acid chains and rapidly fold into compact active structures. Rapid folding is a prerequisite for the survival of a protein in the cell. It is not surprising then that critical steps in the folding process are assisted in the cell. One of the rate-limiting steps in protein folding is the formation of native disulfide bonds. In the cell, they form much more rapidly than in the test tube, implying that a catalyst is present in vivo (1-3).We and others have shown that there is a 21-kDa enzyme called DsbA that is essential for disulfide bond formation in vivo (4, 5). DsbA is a protein-folding catalyst that acts to form disulfides in newly synthesized periplasmic proteins. DsbA acts as the direct donor of disulfides to secreted proteins. The active site of DsbA consists of a pair of cysteines present in a CXXC motif that can oxidize to form a very reactive disulfide (6). This disulfide is rapidly transferred to proteins that are in the process of folding (7,8). The ease at which DsbA can be purified and manipulated biochemically and the availability of a 1.7-Å resolution crystal structure for DsbA have led to an abundance of information concerning the mechanism of DsbA action (9 -16). One of the features that is thought to contribute to the extreme oxidizing power of DsbA is the very low pK a of its Cys-30 residue, which makes it a superb leaving group in disulfide exchange reactions (13-16).In order for DsbA to act as a catalyst of disulfide bond formation, it needs to be reoxidized. Genetic studies strongly implicate an inner membrane protein called DsbB in the reoxidation of DsbA (17,18). Evidence that DsbB is required for the reoxidation of DsbA includes the observation that DsbA accumulates in a reduced form in DsbB mutants (18). The isolation from cells of a DsbA-DsbB dimer covalently linked by a disulfide bond implies a direct interaction between t...

DsbB Elicits a Red-shift of Bound Ubiquinone during the Catalysis of DsbA Oxidation

“…Cells were grown at 37°C on L-medium containing 100 g/ml of ampicillin, induced with 10 M isopropyl-1-thio-␤-D-galactopyranoside at a turbidity of Klett unit 60, and harvested at 4 h after the induction. DsbB and its derivatives were purified essentially as described by Bader et al (13,24). UV spectrum measurements after reduction with sodium borohydride (14) indicated that the purified preparations of DsbB contained ϳ0.5 mol equivalent of endogenously bound UQ.…”

Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

“…Oxidation of the Reduced Variants by DsbB in Vitro-Membranes containing DsbB, ubiquinone, and terminal oxidases were prepared from E. coli JCB819 cells overexpressing DsbB as described (3,35). The DsbB-catalyzed oxidation of reduced DsbA was followed by the decrease in specific DsbA fluorescence at 330 nm (excitation at 280 nm).…”

“…DsbA-mediated disulfide bond formation in the periplasm involves oxidation of reduced, newly translocated substrate polypeptides by DsbA, followed by reoxidation of DsbA by the inner membrane protein DsbB. DsbB is in turn reoxidized by molecular oxygen through ubiquinone and terminal cytochrome oxidases (3,4).…”

Characterization of the Menaquinone-dependent Disulfide Bond Formation Pathway of Escherichia coli

“…For kinetic studies, oxidation of DsbA was determined by the decrease in DsbA fluorescence (25) essentially as described by Bader et al (26). The initial velocity of the reaction was determined from the initial slope of the fluorescence change at 330 nm using a Hitachi F-4500 fluorescence spectrophotometer.…”