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Title
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Intersubunit salt bridges with a sulfate anion control subunit dissociation and thermal stabilization of Bacillus sp. TB-90 urate oxidase.
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Authors
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T.Hibi,
Y.Hayashi,
H.Fukada,
T.Itoh,
T.Nago,
Y.Nishiya.
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Ref.
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Biochemistry, 2014,
53,
3879-3888.
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PubMed id
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Abstract
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The optimal activity of Bacillus sp. TB-90 urate oxidase (BTUO) is 45 °C, but
this enzyme is one of the most thermostable urate oxidases. A marked increase
(>10 °C) in its thermal stability is induced by high concentrations
(0.8–1.2 M) of sodium sulfate. Calorimetric measurements and size exclusion
chromatographic analyses suggested that sulfate-induced thermal stabilization is
related to the binding of a sulfate anion that repressed the dissociation of
BTUO tetramers into dimers. To determine the sulfate binding site, the crystal
structure was determined at 1.75 Å resolution. The bound sulfate anion was
found at the subunit interface of the symmetrical related subunits and formed a
salt bridge with two Arg298 residues in the flexible loop that is involved in
subunit assembly. Site-directed mutagenesis of Arg298 to Glu was used to
extensively characterize the sulfate binding site at the subunit interface. The
network of charged hydrogen bonds via the bound sulfate is suggested to
contribute significantly to the thermal stabilization of both subunit dimers and
the tetrameric assembly of BTUO. Knowledge of the mechanism of salt-induced
stabilization will help to develop new strategies for enhancing protein thermal
stabilization.
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