A staphylococcal capsule-producing enzyme that unfolds via multiple intermediates predominantly exists as the trimers at low concentrations.

CapG, an enzyme expressed by , catalyzes an epimerization reaction to synthesize -acetyl-L-fucosamine, a constituent of capsule involved in pathogenesis. This protein has two domains, exists as the homohexamers in the solution, and usually produces products at hundred-nanomolar concentrations. To determine the folding-unfolding mechanism and the oligomeric form of CapG, particularly at low concentrations, we have investigated a recombinant CapG (rCapG) using different probes. The results show that rCapG in the aqueous solution is well-structured and exists as a mixture of different homo-oligomers such as dimer, trimer, tetramer, and hexamer. A considerable amount of rCapG also remained as the monomers at 0.5-5 µM concentrations. However, its trimeric forms are predominant at 5-100 µM concentrations. The formation of trimers is induced at higher concentrations of rCapG. Besides, rCapG at 0-7 M urea was reversibly unfolded by forming three structurally dissimilar intermediates. The first intermediate was possibly formed by the partial disruption of native rCapG trimers to dimers and monomers, whereas the second intermediate was likely produced due to the swelling and additional dissociation of the first intermediate. Further dissociation/swelling may have generated a third intermediate from the second intermediate. Additionally, both domains of rCapG started unfolding at the same urea concentrations. However, its C-terminal domain mostly completed unfolding at 7 M urea. Collectively, the study has provided new clues about the oligomeric state and the folding mechanism of CapG and also set up a foundation for discovering new anti-CapG molecules in the future.