Cloned (Comment) | Organism |
---|---|
gene Xyl1, phylogenetic analysis, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)-T1R | Scheffersomyces stipitis |
Crystallization (Comment) | Organism |
---|---|
purified enzyme in apoform and in complex with NADPH, X-ray diffraction structure determination and analysis at 1.95-2.0 A resolution, the apoform structure is determined by molecular replacement using the structure of xylose reductase from Candida tenuis (CtXR, PDB ID 1Z9A) as a search model, the structure of SsXR in complex with NADPH is determined by molecular replacement using the crystal structure of the apoform of SsXR (PDB ID 5Z6U), model building | Scheffersomyces stipitis |
Protein Variants | Comment | Organism |
---|---|---|
D47A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
F111A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
F128A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
F221A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
H110A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
L224A | site-directed mutagenesis of the substrate binding residue, the mutant shows 35% XR activity compared to the wild-type enzyme | Scheffersomyces stipitis |
N306A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
W20A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
W311A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
W79A | site-directed mutagenesis of the substrate binding residue, the mutant shows almost complete loss of activity | Scheffersomyces stipitis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.00928 | - |
NADPH | without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
0.0187 | - |
NADH | without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
0.0277 | - |
NADPH | with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
0.136 | - |
NADH | with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
32.37 | - |
D-xylose | with NADPH, without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
39.4 | - |
D-xylose | with NADPH, with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
39.61 | - |
D-xylose | with NADH, without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
59.72 | - |
D-xylose | with NADH, with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
75000 | - |
recombinant His-tagged enzyme, gel filtration | Scheffersomyces stipitis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-xylose + NADH + H+ | Scheffersomyces stipitis | - |
xylitol + NAD+ | - |
r | |
D-xylose + NADH + H+ | Scheffersomyces stipitis NRRL Y-11545 | - |
xylitol + NAD+ | - |
r | |
D-xylose + NADH + H+ | Scheffersomyces stipitis NBRC 10063 | - |
xylitol + NAD+ | - |
r | |
D-xylose + NADH + H+ | Scheffersomyces stipitis ATCC 58785 | - |
xylitol + NAD+ | - |
r | |
D-xylose + NADPH + H+ | Scheffersomyces stipitis | - |
xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | Scheffersomyces stipitis NRRL Y-11545 | - |
xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | Scheffersomyces stipitis NBRC 10063 | - |
xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | Scheffersomyces stipitis ATCC 58785 | - |
xylitol + NADP+ | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Scheffersomyces stipitis | P31867 | - |
- |
Scheffersomyces stipitis ATCC 58785 | P31867 | - |
- |
Scheffersomyces stipitis NBRC 10063 | P31867 | - |
- |
Scheffersomyces stipitis NRRL Y-11545 | P31867 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)-T1R by nickel affinity chromatography and gel filtration | Scheffersomyces stipitis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
D-xylose + NADH + H+ | - |
Scheffersomyces stipitis | xylitol + NAD+ | - |
r | |
D-xylose + NADH + H+ | - |
Scheffersomyces stipitis NRRL Y-11545 | xylitol + NAD+ | - |
r | |
D-xylose + NADH + H+ | - |
Scheffersomyces stipitis NBRC 10063 | xylitol + NAD+ | - |
r | |
D-xylose + NADH + H+ | - |
Scheffersomyces stipitis ATCC 58785 | xylitol + NAD+ | - |
r | |
D-xylose + NADPH + H+ | - |
Scheffersomyces stipitis | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | D-xylose binding mode of SsXR is elucidated by molecular docking simulations of SsXR with the D-xylose substrate revealing that D-xylose fits well into the predicted substrate binding pocket. The D-xylose binding pocket consists of 10 residues: Trp20, Asp47, Trp79, His110, Phe111, Phe128, Phe221, Leu224, Asn306, and Trp311. The Asp47 residue contributes to the stabilization of two hydroxyl groups (OH2 and OH3), and the aldehyde group of D-xylose is stabilized by Asn306 through hydrogen bonding. The residues involved in formation of the D-xylose binding pocket are confirmed by site-directed mutagenesis experiments | Scheffersomyces stipitis | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | - |
Scheffersomyces stipitis NRRL Y-11545 | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | D-xylose binding mode of SsXR is elucidated by molecular docking simulations of SsXR with the D-xylose substrate revealing that D-xylose fits well into the predicted substrate binding pocket. The D-xylose binding pocket consists of 10 residues: Trp20, Asp47, Trp79, His110, Phe111, Phe128, Phe221, Leu224, Asn306, and Trp311. The Asp47 residue contributes to the stabilization of two hydroxyl groups (OH2 and OH3), and the aldehyde group of D-xylose is stabilized by Asn306 through hydrogen bonding. The residues involved in formation of the D-xylose binding pocket are confirmed by site-directed mutagenesis experiments | Scheffersomyces stipitis NRRL Y-11545 | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | - |
Scheffersomyces stipitis NBRC 10063 | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | D-xylose binding mode of SsXR is elucidated by molecular docking simulations of SsXR with the D-xylose substrate revealing that D-xylose fits well into the predicted substrate binding pocket. The D-xylose binding pocket consists of 10 residues: Trp20, Asp47, Trp79, His110, Phe111, Phe128, Phe221, Leu224, Asn306, and Trp311. The Asp47 residue contributes to the stabilization of two hydroxyl groups (OH2 and OH3), and the aldehyde group of D-xylose is stabilized by Asn306 through hydrogen bonding. The residues involved in formation of the D-xylose binding pocket are confirmed by site-directed mutagenesis experiments | Scheffersomyces stipitis NBRC 10063 | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | - |
Scheffersomyces stipitis ATCC 58785 | xylitol + NADP+ | - |
r | |
D-xylose + NADPH + H+ | D-xylose binding mode of SsXR is elucidated by molecular docking simulations of SsXR with the D-xylose substrate revealing that D-xylose fits well into the predicted substrate binding pocket. The D-xylose binding pocket consists of 10 residues: Trp20, Asp47, Trp79, His110, Phe111, Phe128, Phe221, Leu224, Asn306, and Trp311. The Asp47 residue contributes to the stabilization of two hydroxyl groups (OH2 and OH3), and the aldehyde group of D-xylose is stabilized by Asn306 through hydrogen bonding. The residues involved in formation of the D-xylose binding pocket are confirmed by site-directed mutagenesis experiments | Scheffersomyces stipitis ATCC 58785 | xylitol + NADP+ | - |
r | |
additional information | the bottleneck of the enzyme activity in SsXR appears to be the binding affinity for D-xylose | Scheffersomyces stipitis | ? | - |
- |
|
additional information | the bottleneck of the enzyme activity in SsXR appears to be the binding affinity for D-xylose | Scheffersomyces stipitis NRRL Y-11545 | ? | - |
- |
|
additional information | the bottleneck of the enzyme activity in SsXR appears to be the binding affinity for D-xylose | Scheffersomyces stipitis NBRC 10063 | ? | - |
- |
|
additional information | the bottleneck of the enzyme activity in SsXR appears to be the binding affinity for D-xylose | Scheffersomyces stipitis ATCC 58785 | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
monomer or dimer | x * 37500, about | Scheffersomyces stipitis |
More | two SsXR polypeptide chains in an asymmetric unit form a dimer. SsXR generally separated into monomers in 50 and 100 mM NaCl and completely separated into monomers in150 mM NaCl, although it exists as dimers in the absence of NaCl. Based on these results, it is suggested that SsXR exists as a monomer under the physiological NaCl concentration and tends to form a dimer in the presence of low NaCl concentrations. Oligomer formation affects enzyme activity. The monomeric structure of SsXR is composed of 15 alpha-helices (alpha1-alpha15) and 10 beta-strands (beta1-beta10). The monomeric structure of SsXR consists of a core domain and two auxiliary regions (ARs), AR-I and AR-II. The core domain consists of 13 alpha-helices (alpha1-alpha10, alpha12-alpha13, and alpha15) and eight beta-strands (beta3-beta10) and forms a TIM-barrel motif. As the conventional TIM-barrel motif, in SsXR eight parallel beta-strands (beta3-beta10) are arranged in a cylindrical shape with eight surrounding alpha-helices (alpha1, alpha3, alpha5-alpha8, and alpha12-alpha13). Four alpha-helices (alpha2, alpha9-alpha10, and alpha15) are located at the back of the TIM-barrel motif and contribute to binding of the NADPH cofactor. AR-I is composed of two beta-strands (beta1-beta2) and is located on the opposite side of the TIM-barrel. AR-II consists of two alpha-helices (alpha11 and alpha14) and is positioned next to the alpha12 helix. As reported in other enzymes belonging to AKR families, four catalytic residues, Asp43, Tyr48, Lys77, and His110, are also conserved in the SsXR, and involved in the catalytic mechanism | Scheffersomyces stipitis |
Synonyms | Comment | Organism |
---|---|---|
SsXR | - |
Scheffersomyces stipitis |
XYL1 | - |
Scheffersomyces stipitis |
xylose reductase | - |
Scheffersomyces stipitis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Scheffersomyces stipitis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.39 | - |
NADH | with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
2.68 | - |
NADH | without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
3.02 | - |
D-xylose | with NADH, with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
4.34 | - |
D-xylose | with NADH, without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
6.69 | - |
D-xylose | with NADPH, with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
7.63 | - |
NADPH | with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
7.65 | - |
NADPH | without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
8.37 | - |
D-xylose | with NADPH, without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Scheffersomyces stipitis |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
NAD+ | - |
Scheffersomyces stipitis | |
NADH | - |
Scheffersomyces stipitis | |
NADP+ | - |
Scheffersomyces stipitis | |
NADPH | the enzyme protein undergoes an open/closed conformation change upon NADPH binding, SsXR structure in complex with the NADPH cofactor, overview | Scheffersomyces stipitis |
General Information | Comment | Organism |
---|---|---|
evolution | phylogenetic tree analysis, overview | Scheffersomyces stipitis |
additional information | structure-function analysis, molecular docking simulation, overview. The SsXR structure in complex with the NADPH cofactor shows that the protein undergoes an open/closed conformation change upon NADPH binding. The substrate binding pocket of SsXR is somewhat hydrophobic, which seems to result in low binding affinity to the substrate. The monomeric structure of SsXR is composed of 15 alpha-helices (alpha1-alpha15) and 10 beta-strands (beta1-beta10). The monomeric structure of SsXR consists of a core domain and two auxiliary regions (ARs), AR-I and AR-II. The core domain consists of 13 alpha-helices (alpha1-alpha10, alpha12-alpha13, and alpha15) and eight beta-strands (beta3-beta10) and forms a TIM-barrel motif. As the conventional TIM-barrel motif, in SsXR eight parallel beta-strands (beta3-beta10) are arranged in a cylindrical shape with eight surrounding alpha-helices (alpha1, alpha3, alpha5-alpha8, and alpha12-alpha13). Four alpha-helices (alpha2, alpha9-alpha10, and alpha15) are located at the back of the TIM-barrel motif and contribute to binding of the NADPH cofactor. AR-I is composed of two beta-strands (beta1-beta2) and is located on the opposite side of the TIM-barrel. AR-II consists of two alpha-helices (alpha11 and alpha14) and is positioned next to the alpha12 helix. As reported in other enzymes belonging to AKR families, four catalytic residues, Asp43, Tyr48, Lys77, and His110, are also conserved in the SsXR, and involved in the catalytic mechanism | Scheffersomyces stipitis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.051 | - |
D-xylose | with NADH, with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
0.11 | - |
D-xylose | with NADH, without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
0.17 | - |
D-xylose | with NADPH, with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
0.259 | - |
D-xylose | with NADPH, without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
17.57 | - |
NADH | with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
143.3 | - |
NADH | without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
275.5 | - |
NADPH | with 150 mM NaCl, pH 8.0, 30°C | Scheffersomyces stipitis | |
824.4 | - |
NADPH | without NaCl, pH 8.0, 30°C | Scheffersomyces stipitis |