The cofactors and domains of a staphylococcal capsule-producing enzyme preserve its structure, stability, shape, and dimerization ability.

CapF, a staphylococcal capsule-producing enzyme, binds Zn2+ ion and NADPH using its C-terminal domain (CTD) and N-terminal domain (NTD), respectively. To elucidate the roles of cofactors and domains, we have systematically investigated the related recombinant proteins, rCapF, rCTD, rNTD, and the Zn2+-free rCapF/rCTD, Apo-rCapF/Apo-rCTD. The results show that the secondary structure, tertiary structure, shape, and surface hydrophobicity of Apo-rCapF and Apo-rCTD are different from those of rCapF and rCTD. The removal of Zn2+ made rCapF thermo-sensitive, whereas both rCTD and Apo-rCTD are thermo-resistant proteins. Further, Apo-rCapF and rCapF existed as the dimers, whereas rCTD and Apo-rCTD formed a mixture of dimers and tetramers in the aqueous solution. Zn2+ maintained the structure of NTD as well. The NADPH binding activity and Cys accessibility of rNTD, rCapF, and Apo-rCapF were significantly different from each other. The binding of NADPH to the above three proteins freely occurred, liberated heat at 250C, and increased their diameters. In addition, the structure, stability, shape, and oligomerization ability of rNTD, rCTD, and rCapF little resembled each other. Collectively, the domains and cofactors of CapF contribute to preserving its conformation, stability, shape, and dimerization ability.