Roles of a conserved arginine residue of DsbB in linking protein disulfide-bond-formation pathway to the respiratory chain of Escherichia coli.
The active-site cysteines of DsbA, the periplasmic disulfide-bond-forming enzyme of Escherichia coli, are kept oxidized by the cytoplasmic membrane protein DsbB. DsbB, in turn, is oxidized by two kinds of quinones (ubiquinone for aerobic and menaquinone for anaerobic growth) in the electron-transport chain. We describe the isolation of dsbB missense mutations that change a highly conserved arginine residue at position 48 to histidine or cysteine. In these mutants, DsbB functions reasonably well aerobically but poorly anaerobically. Consistent with this conditional phenotype, purified R48H exhibits very low activity with menaquinone and an apparent Michaelis constant (K(m)) for ubiquinone seven times greater than that of the wild-type DsbB, while keeping an apparent K(m) for DsbA similar to that of wild-type enzyme. From these results, we propose that this highly conserved arginine residue of DsbB plays an important role in the catalysis of disulfide bond formation through its role in the interaction of DsbB with quinones.
Kadokura
H
H
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
Bader
M
M
Tian
H
H
Bardwell
J C
JC
Beckwith
J
J
D000595:Amino Acid Sequence; D001120:Arginine; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004579:Electron Transport / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D001120:Arginine; D010088:Oxidoreductases; D019704:Protein Disulfide-Isomerases