Mutational analysis of the disulfide catalysts DsbA and DsbB.
In prokaryotes, disulfides are generated by the DsbA-DsbB system. DsbB functions to generate disulfides by quinone reduction. These disulfides are passed to the DsbA protein and then to folding proteins. To investigate the DsbA-DsbB catalytic system, we performed an in vivo selection for chromosomal dsbA and dsbB mutants. We rediscovered many residues previously shown to be important for the activity of these proteins. In addition, we obtained one novel DsbA mutant (M153R) and four novel DsbB mutants (L43P, H91Y, R133C, and L146R). We also mutated residues that are highly conserved within the DsbB family in an effort to identify residues important for DsbB function. We found classes of mutants that specifically affect the apparent K(m) of DsbB for either DsbA or quinones, suggesting that quinone and DsbA may interact with different regions of the DsbB protein. Our results are consistent with the interpretation that the residues Q33 and Y46 of DsbB interact with DsbA, Q95 and R48 interact with quinones, and that residue M153 of DsbA interacts with DsbB. All of these interactions could be due to direct amino acid interactions or could be indirect through, for instance, their effect on protein structure. In addition, we find that the DsbB H91Y mutant severely affects the k(cat) of the reaction between DsbA and DsbB and that the DsbB L43P mutant is inactive, suggesting that both L43 and H91 are important for the activity of DsbB. These experiments help to better define the residues important for the function of these two protein-folding catalysts.
Tan
Jacqueline
J
Department of Molecular and Cellular Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.
Lu
Ying
Y
Bardwell
James C A
JC
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D025941:Protein Interaction Mapping; D011809:Quinones / Q000378:metabolism
D001426:Bacterial Proteins; C079503:DsbB protein, Bacteria; D008565:Membrane Proteins; D011809:Quinones; D019704:Protein Disulfide-Isomerases