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