Engineered pathways for correct disulfide bond oxidation.
Correct formation of disulfide bonds is critical for protein folding. We find that cells lacking protein disulfide isomerases (PDIs) can use alternative mechanisms for correct disulfide bond formation. By linking correct disulfide bond formation to antibiotic resistance, we selected mutants that catalyze correct disulfide formation in the absence of DsbC, Escherichia coli's PDI. Most of our mutants massively overproduce the disulfide oxidase DsbA and change its redox status. They enhance DsbA's ability to directly form the correct disulfides by increasing the level of mixed disulfides between DsbA and substrate proteins. One mutant operates via a different mechanism; it contains mutations in DsbB and CpxR that alter the redox environment of the periplasm and increases the level of the chaperone/protease DegP, allowing DsbA to gain disulfide isomerase ability in vivo. Thus, given the proper expression level, redox status, and chaperone assistance, the oxidase DsbA can readily function in vivo to catalyze the folding of proteins with complex disulfide bond connectivities. Our selection reveals versatile strategies for correct disulfide formation in vivo. Remarkably, our evolution of new pathways for correct disulfide bond formation in E. coli mimics eukaryotic PDI, a highly abundant partially reduced protein with chaperone activity.
Department of Molecular, Cellular, and Developmental Biology, Howard Hughes Medical Institute, University of Michigan, 830 N. University Ave., Ann Arbor, MI 48109-1048, USA.
Guoping
G
Ren
James C A
JC
Bardwell
D004220:Disulfides / Q000737:chemistry*; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D033903:Periplasmic Proteins / Q000378:metabolism; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D012697:Serine Endopeptidases / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins; D033903:Periplasmic Proteins; C061682:DegP protease; D012697:Serine Endopeptidases; D001618:beta-Lactamases; D019704:Protein Disulfide-Isomerases; C526649:dsbC protein, E coli
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