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Literature summary for 1.1.1.431 extracted from

  • Son, H.F.; Lee, S.M.; Kim, K.J.
    Structural insight into D-xylose utilization by xylose reductase from Scheffersomyces stipitis (2018), Sci. Rep., 8, 17442 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

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 (Commentary)

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

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 [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 [Da] Molecular Weight Maximum [Da] Comment Organism
75000
-
recombinant His-tagged enzyme, gel filtration Scheffersomyces stipitis

Natural Substrates/ Products (Substrates)

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

Organism UniProt Comment Textmining
Scheffersomyces stipitis P31867
-
-
Scheffersomyces stipitis ATCC 58785 P31867
-
-
Scheffersomyces stipitis NBRC 10063 P31867
-
-
Scheffersomyces stipitis NRRL Y-11545 P31867
-
-

Purification (Commentary)

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 and Products (Substrate)

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

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

Synonyms Comment Organism
SsXR
-
Scheffersomyces stipitis
XYL1
-
Scheffersomyces stipitis
xylose reductase
-
Scheffersomyces stipitis

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
assay at Scheffersomyces stipitis

Turnover Number [1/s]

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

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Scheffersomyces stipitis

Cofactor

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

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 [mM/s]

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