Effect of sequences of the active-site dipeptides of DsbA and DsbC on in vivo folding of multidisulfide proteins in Escherichia coli

We have examined the role of the active-site CXXC central dipeptides of Dsb A and DsbC in disulfide bond formation and isomerization in the Escherichia coli periplasm, DsbA active-site mutants with a wide range elf redox poten tials were expressed either from the trc promoter on a multicopy plasmid or from the endogenous dsb/A promoter by integration of the respective allele s into the bacterial chromosome. The dsbA alleles gave significant differen ces in the yield of active murine urokinase, a protein containing 12 disulf ides, including some that significantly enhanced urokinase expression over that allowed by wild-type DsbA. No direct correlation between the in vitro redox potential of dsbA variants and the urokinase yield was observed, Thes e results suggest that the active-site CXXC motif of DsbA. can play an impo rtant role in determining the folding of multidisulfide proteins, in a way that is independent from DsbA's redox potential. However, under aerobic con ditions, there was no significant difference among the DsbA mutants with re spect to phenotypes depending on the oxidation of proteins with few disulfi de bonds. The effect of active-site mutations in the CXXC motif of DsbC on disulfide isomerization in vivo was also examined, A library of DsbC expres sion plasmids with the active-site dipeptide randomized was screened for mu tants that have increased disulfide isomerization activity. A number of Dsb C mutants that showed enhanced expression of a variant of human tissue plas minogen activator as well as mouse urokinase were obtained. These DsbC muta nts overwhelmingly contained an aromatic residue at the C-terminal position of the dipeptide, whereas the N-terminal residue was more diverse. Collect ively, these data indicate that the active sites of the soluble thiol- disu lfide oxidoreductases can be modulated to enhance disulfide isomerization a nd protein folding in the bacterial periplasmic space.