Conversion of a catalytic into a structural disulfide bond by circular permutation

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• DsbA from Escherichia coli is a potent thiol-disulfide oxidoreductase essential for disulfide bond formation in the bacterial periplasm.
• Its catalytic domain features a thioredoxin-like fold with a Cys30-Pro31-His32-Cys33 active site disulfide bridge.
• The wild-type DsbA disulfide bond is destabilized by the low pKa of Cys30, facilitating its oxidative activity.

Purpose of the Study:

• To characterize a circularly permuted DsbA variant (H32-P31) with altered termini.
• To investigate the structural and functional consequences of circular permutation on DsbA's catalytic activity and disulfide bond properties.

Main Methods:

• Construction and characterization of a circularly permuted DsbA variant (H32-P31).
• Structural analysis of the variant.
• Assessment of folding properties in both redox states.
• In vitro and in vivo assays for dithiol oxidase activity.
• Determination of cysteine thiol pKa values and redox potential.

Main Results:

• The H32-P31 variant adopts a wild-type-like structure and exhibits reversible, cooperative folding.
• Despite structural similarity, H32-P31 is catalytically inactive as a dithiol oxidase.
• The variant displays significantly higher pKa values for both cysteine thiols (> 8).
• H32-P31 is 500-fold more reducing than wild-type DsbA and more stable in its oxidized form.

Conclusions:

• Circular permutation of DsbA alters the properties of the active site disulfide bond.
• The disulfide bond in H32-P31 behaves as a structural disulfide rather than a catalytic one.
• This suggests that the precise positioning and environment of the active site cysteines are critical for DsbA's oxidative function.