5AYJ

Hyperthermostable mutant of Bacillus sp. TB-90 Urate Oxidase - R298C

Summary for 5AYJ

• Entry DOI: 10.2210/pdb5ayj/pdb
• Related: 1J2G 3WLV
• Descriptor: Uric acid degradation bifunctional protein, 9-METHYL URIC ACID, HEXAETHYLENE GLYCOL, ... (5 entities in total)
• Functional Keywords: disulfide bond, thermostability, protein engineering, oxidase, subunit-subunit interaction, oxidoreductase
• Biological source: Bacillus sp. TB-90
• Total number of polymer chains: 4
• Total formula weight: 154229.16

Authors

Hibi, T.,Kume, A.,Kawamura, A.,Itoh, T.,Nishiya, Y. (deposition date: 2015-08-21, release date: 2016-01-20, Last modification date: 2024-10-16)

Primary citation

Hibi, T.,Kume, A.,Kawamura, A.,Itoh, T.,Fukada, H.,Nishiya, Y.
Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity
Biochemistry, 55:724-732, 2016

PubMed Abstract: Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi, T., et al. (2014) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298 residues, which stabilized the packing between two β-barrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by ∼ 20 °C compared with that of the wild-type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppression of the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cβ distance between Arg298 residues of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide cross-link. Disulfide bonding was associated with local disordering of interface loop II (residues 277-300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the C-terminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help in the development of new strategies for enhancing the thermostabilization of multimeric proteins.

PubMed: 26739254
DOI: 10.1021/acs.biochem.5b01119

Experimental method

X-RAY DIFFRACTION (2.05 Å)