3 research outputs found
Construction of Thermophilic Xylanase and Its Structural Analysis
The
glycoside hydrolase family 11 xylanase has been utilized in
a wide variety of industrial applications, from food processing to
kraft pulp bleaching. Thermostability enhances the economic value
of industrial enzymes by making them more robust. Recently, we determined
the crystal structure of an endo-β-1,4-xylanase (GH11) from
mesophilic <i>Talaromyces cellulolyticus</i>, named XylC.
Ligand-free XylC exists to two conformations (open and closed forms).
We found that the âclosedâ structure possessed an unstable
region within the N-terminal region far from the active site. In this
study, we designed the thermostable xylanase by the structure-based
site-directed mutagenesis on the N-terminal region. In total, nine
mutations (S35C, N44H, Y61M, T62C, N63L, D65P, N66G, T101P, and S102N)
and an introduced disulfide bond of the enzyme contributed to the
improvement in thermostability. By combining the mutations, we succeeded
in constructing a mutant for which the melting temperature was partially
additively increased by >20 °C (measured by differential scanning
calorimetry) and the activity was additively enhanced at elevated
temperatures, without loss of the original specific activity. The
crystal structure of the most thermostable mutant was determined at
2.0 Ă
resolution to elucidate the structural basis of thermostability.
From the crystal structure of the mutant, it was revealed that the
formation of a disulfide bond induces new CâC contacts and
a conformational change in the N-terminus. The resulting induced conformational
change in the N-terminus is key for stabilizing this region and for
constructing thermostable mutants without compromising the activity
Hyperstabilization of Tetrameric <i>Bacillus</i> sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity
<i>Bacillus</i> sp. TB-90 urate oxidase (BTUO) is one
of the most thermostable homotetrameric enzymes. We previously reported
[Hibi, T., et al. (2014) <i>Biochemistry</i> <i>53</i>, 3879â3888] 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 âź20 °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 Ă
resolution revealed distinct disulfide bond formation between the
symmetrically related subunits via Cys298, although the C<sub>β</sub> distance between Arg298 residues of the wild-type enzyme (5.4 Ă
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 277â300), 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
Seven Cysteine-Deficient Mutants Depict the Interplay between Thermal and Chemical Stabilities of Individual Cysteine Residues in Mitogen-Activated Protein Kinase câJun NâTerminal Kinase 1
Intracellular proteins can have free cysteines that may
contribute to their structure, function, and stability; however, free
cysteines can lead to chemical instabilities in solution because of
oxidation-driven aggregation. The MAP kinase, c-Jun N-terminal kinase
1 (JNK1), possesses seven free cysteines and is an important drug
target for autoimmune diseases, cancers, and apoptosis-related diseases.
To characterize the role of cysteine residues in the structure, function,
and stability of JNK1, we prepared and evaluated wild-type JNK1 and
seven cysteine-deficient JNK1 proteins. The nonreduced sodium dodecyl
sulfateâpolyacrylamide gel electrophoresis experiments showed
that the chemical stability of JNK1 increased as the number of cysteines
decreased. The contribution of each cysteine residue to biological
function and thermal stability was highly susceptible to the environment
surrounding the particular cysteine mutation. The mutations of solvent-exposed
cysteine to serine did not influence biological function and increased
the thermal stability. The mutation of the accessible cysteine involved
in the hydrophobic pocket did not affect biological function, although
a moderate thermal destabilization was observed. Cysteines in the
loosely
assembled hydrophobic environment moderately contributed to thermal
stability, and the mutations of these cysteines had a negligible effect
on enzyme activity. The other cysteines are involved in the tightly
filled hydrophobic core, and mutation of these residues was found
to correlate with thermal stability and enzyme activity. These findings
about the role of cysteine residues should allow us to obtain a stable
JNK1 and thus promote the discovery of potent JNK1 inhibitors