De novo design and evolution of artificial disulfide isomerase enzymes analogous to the bacterial DsbC

Abstract

The Escherichia coli disulfide isomerase, DsbC is a V-shaped homodimer with each monomer comprising a dimerization region that forms part of a putative peptide-binding pocket and a thioredoxin catalytic domain. Disulfide isomerases from prokaryotes and eukaryotes exhibit little sequence homology but display very similar structural organization with two thioredoxin domains facing each other on top of the dimerization/peptide-binding region. To aid the understanding of the mechanistic significance of thioredoxin domain dimerization and of the peptide-binding cleft of DsbC, we constructed a series of protein chimeras comprising unrelated protein dimerization domains fused to thioredoxin superfamily enzymes. Chimeras consisting of the dimerization domain and the alpha-helical linker of the bacterial proline cis/trans isomerase FkpA and the periplasmic oxidase DsbA gave rise to enzymes that catalyzed the folding of multidisulfide substrate proteins in vivo with comparable efficiency to E. coli DsbC. In addition, expression of FkpA-DsbAs conferred modest resistance to CuCl2, a phenotype that depends on disulfide bond isomerization. Selection for resistance to elevated CuCl2 concentrations led to the isolation of FkpA-DsbA mutants containing a single amino acid substitution that changed the active site of the DsbA domain from CPHC into CPYC, increasing the similarity to the DsbC active site (CGYC). Unlike DsbC, which is resistant to oxidation by DsbB-DsbA and does not normally catalyze disulfide bond formation under physiological conditions, the FkpA-DsbA chimeras functioned both as oxidases and isomerases. The engineering of these efficient artificial isomerases delineates the key features of catalysis of disulfide bond isomerization and enhances our understanding of its evolution.

FIGURE 1.

A, structures of DsbC, DsbG, and PDI exhibit similar domain arrangement. B, protein structures of DsbA and FkpA, and a predictive molecular model of FkpA-DsbA2. The amino acid sequence at the fusion region of the two proteins is shown.

FIGURE 2.

Disulfide-bond formation in vivo. A, yield of active vtPA in dsbC (gray bars) or dsbA (black bars) cells. E. coli PB351 and PB401 (respectively) were transformed with pTrcStIIvtPA and pBAD derivatives encoding the respective FkpA-DsbA fusion proteins and grown in LB media. Protein synthesis was induced as described under “Experimental Procedures,” and the yield of active vtPA was determined 3 h after induction. Relative activities were obtained by dividing the ΔA₄₀₅ of each strain (subtracted of the background consisting of a strain not expressing vtPA) by the ΔA₄₀₅ of a strain expressing vtPA alone. B, PhoA activity in E. coli MC1000 dsbA (white bars) and MC1000 dsbB (black bars). The alkaline phosphatase activity in the parental isogenic strain MC1000 is shown by the gray bar.

FIGURE 3.

FkpA-DsbA2 mutants obtained following mutagenesis and selection for CuCl₂ resistance. A, molecular model of the FkpA-DsbA2m18 mutant. B, yield of active vtPA in E. coli PB351 (SF100 ΔdsbC) obtained as described in Fig. 2A. C, AppA activity assayed as described under “Experimental Procedures.” The AppA activity was determined by measuring A₄₁₀. One unit was defined as 1,000 × A₄₁₀ per min/ml (4).