The effects of free Cys residues on the structure, activity, and tetrameric stability of mammalian uricase.
Appl Microbiol Biotechnol
; 107(14): 4533-4542, 2023 Jul.
Article
em En
| MEDLINE
| ID: mdl-37256327
ABSTRACT
Mammalian uricases contain four conserved cysteine (Cys) residues, but little is known about their structures and functions. In this study, we first confirmed that all four Cys residues are free and not involved in disulfide bond formation, using canine uricase as a model protein. Cys residues had a greater effect on stability than on activity based on single Cys-to-Ser (serine) substitutions. Circular dichroism (CD) and homology modeling indicated that C188S reduces ß-sheet contents and inter- and intra-subunit hydrophobic interaction, potentially impairing the core tetrameric ß-barrel structure of the tunneling-fold protein, and ultimately decreased the tetrameric stability. Additionally, the inactivation of C188S during the stability tests may be a complex process involving depolymerization followed by irregular aggregation. Double mutations or thiol blockage of Cys188 and Cys195 significantly disrupted the formation and stability of tetrameric uricase, which may be mediated by the free thiols in Cys residues. The present results demonstrated that the free Cys residues are essential for tetrameric formation and stability in mammalian uricase. This implies that free cysteine residues, although not involved in disulfide bonding, may play important structural roles in certain proteins, underscoring the significance of the hydrophobic characteristics of the free thiols in Cys residues. KEY POINTS ⢠Four Cys residues are not involved in disulfide bonding in mammalian uricase. ⢠The hydrophobicity of free thiols is critical for tetrameric stability in uricase. ⢠Free Cys residues can serve structural roles without involving in disulfide bonds.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Urato Oxidase
/
Cisteína
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Appl Microbiol Biotechnol
Ano de publicação:
2023
Tipo de documento:
Article