Identification of the ubiquinone-binding domain in the disulfide catalyst disulfide bond protein B.
Disulfide bond (Dsb) formation is catalyzed in the periplasm of prokaryotes by the Dsb proteins. DsbB, a key enzyme in this process, generates disulfides de novo by using the oxidizing power of quinones. To explore the mechanism of this newly described enzymatic activity, we decided to study the ubiquinone-protein interaction and identify the ubiquinone-binding domain in DsbB by cross-linking to photoactivatable quinone analogues. When purified Escherichia coli DsbB was incubated with an azidoubiquinone derivative, 3-azido-2-methyl-5-[(3)H]methoxy-6-decyl-1,4-benzoquinone ([(3)H]azido-Q), and illuminated with long wavelength UV light, the decrease in enzymatic activity correlated with the amount of 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone (azido-Q) incorporated into the protein. One azido-Q-linked peptide with a retention time of 33.5 min was obtained by high performance liquid chromatography of the V8 digest of [(3)H]azido-Q-labeled DsbB. This peptide has a partial NH(2)-terminal amino acid sequence of NH(2)-HTMLQLY corresponding to residues 91-97. This sequence occurs in the second periplasmic domain of the inner membrane protein DsbB in a loop connecting transmembrane helices 3 and 4. We propose that the quinone-binding site is within or very near to this sequence.
Xie
Tong
T
Department of Biochemistry & Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA.
Yu
Linda
L
Bader
Martin W
MW
Bardwell
James C A
JC
Yu
Chang-An
CA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D011485:Protein Binding; D014451:Ubiquinone / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D019920:Photoaffinity Labels; D014451:Ubiquinone