Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

Abstract

In the bacterial periplasm the co-existence of a catalyst of disulfide bond formation (DsbA) that is maintained in an oxidized state and of a reduced enzyme that catalyzes the rearrangement of mispaired cysteine residues (DsbC) is important for the folding of proteins containing multiple disulfide bonds. The kinetic partitioning of the DsbA/DsbB and DsbC/DsbD pathways partly depends on the ability of DsbB to oxidize DsbA at rates >1000 times greater than DsbC. We show that the resistance of DsbC to oxidation by DsbB is abolished by deletions of one or more amino acids within the ␣-helix that connects the N-terminal dimerization domain with the C-terminal thioredoxin domain. As a result, mutant DsbC carrying ␣-helix deletions could catalyze disulfide bond formation and complemented the phenotypes of dsbA cells. Examination of DsbC homologues from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Yersinia pseudotuberculosis, Vibrio cholerae (30 -70% sequence identity with the Escherichia coli enzyme) revealed that the mechanism responsible for avoiding oxidation by DsbB is a general property of DsbC family enzymes. In addition we found that deletions in the linker region reduced, but did not abolish, the ability of DsbC to assist the formation of active vtPA and phytase in vivo, in a DsbD-dependent manner, revealing that interactions between DsbD and DsbC are also conserved.

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

“…3 These data are in agreement with the published studies on PDI reporting that the minimal structure for simple disulfide isomerization includes one catalytic domain (a or a) in combination with the non-catalytic b domain, the latter identified as the primary substrate binding site (22,24). (53), and the experimental evidence showing that monomeric DsbC and ␣-helix deletion mutants of DsbC with re-oriented catalytic domains are able to catalyze DsbB-dependent oxidation (11,12).…”

“…2B). While overexpression of wt DsbC does not restore PhoA activity, the expression of DsbC variants with a perturbed tertiary structure results in PhoA levels Ͼ30% of those in wt cells (12). In contrast, expression of mDsbC-mDsbC conferred only background levels of PhoA activity.…”

“…Single amino acid deletions in the ␣-helical linker that joins the catalytic and the dimerization domains render the protein susceptible to oxidation by DsbB. In turn, oxidized DsbC accumulates in the periplasm where it catalyzes the formation of disulfide bonds in substrate proteins and thus can partially complement dsbA mutants (12). We evaluated whether the covalent linkage of two DsbC monomers might have perturbed the tertiary structure of the molecule to make it susceptible to oxidation.…”

“…Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 10 3 -10 7 -fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11,12).DsbC is a homodimer with each monomer comprising an N-terminal dimerization domain and a C-terminal thioredoxin-like catalytic domain fused by an ␣-helical linker. The crystal structure of DsbC reveals that the two monomers come together to form a V-shaped protein.…”

“…Accordingly, reactions between enzymes of the two pathways, for example the oxidation of DsbC by DsbB or the reduction of DsbA by DsbD, are 10 3 -10 7 -fold slower than the physiologically relevant DsbA-DsbB and DsbC-DsbD reactions (10). Nonetheless, the kinetic barrier between DsbB and DsbC can be breached by introducing mutations that result in structural changes in DsbC (11,12).…”