Role of the cytosolic loop of DsbB in catalytic turnover of the ubiquinone-DsbB complex.
DsbB, an Escherichia coli plasma membrane protein, oxidizes DsbA, the protein dithiol oxidant in the periplasm, in conjunction with respiratory quinone molecules. While its two periplasmic regions, in particular the essential Cys41-Cys44 and the Cys104-Cys130 cysteine pairs, have been characterized in considerable detail, little or no information is available about the functional importance of its three cytosolically disposed regions. In this work the authors introduced insertion and substitution mutations into the short ( approximately 6 residue) central cytosolic loop. The purified mutant proteins proved to have two of the essential cysteines reduced and to exhibit the spectroscopic transition of bound ubiquinone constitutively. A thrombin-cleavage site present in a mutant protein called DsbB-T established that the mutant protein had a rearranged Cys41-Cys130 disulfide that would unpair Cys44. Although this covalent structure of DsbB is reminiscent of the DsbB-DsbA intermediate, in which unpaired Cys44 induces the ubiquinone transition, it is inactive because of the premature disulfide rearrangement without involving DsbA. In addition, ubiquione-mediated in vitro oxidation of reduced DsbB-T was aborted at a half-oxidized state, without rapidly producing the fully oxidized enzyme. Thus, the cytosolic loop alterations compromised the catalytic turnover of DsbB in vitro. These observations suggest that the cytosolic loop is important to coordinate the active-site residues of DsbB and ubiquinone to allow their proper reaction cycles.
Takahashi
Yoh-Hei
YH
Institute for Virus Research, Kyoto University and CREST, Japan Science and Technology Agency, Kyoto, Japan.
Inaba
Kenji
K
Ito
Koreaki
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014451:Ubiquinone / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; C079503:DsbB protein, Bacteria; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D013438:Sulfhydryl Compounds; D014451:Ubiquinone; D003545:Cysteine