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Literature summary extracted from

  • Kratzer, R.; Leitgeb, S.; Wilson, D.K.; Nidetzky, B.
    Probing the substrate binding site of Candida tenuis xylose reductase (AKR2B5) with site-directed mutagenesis (2006), Biochem. J., 393, 51-58.
    View publication on PubMedView publication on EuropePMC

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.431 the purified N309D mutant is crystallized by the hanging-drop vapour-diffusion method at 25°C. The best-diffracting crystals are grown using a well solution consisting of 2.1 M (NH4)2SO4, 100 mM sodium acetate and 100 mM sodium citrate, pH 6.4. Comparison of the 2.4 A X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations Yamadazyma tenuis

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.431 D50A mutant shows 31% and 18% of the wild-type catalytic-centre activities for xylose reduction and xylitol oxidation respectively, consistent with a decrease in the rates of the chemical steps caused by the mutation, but no change in the apparent substrate binding constants and the pattern of substrate specificities Yamadazyma tenuis
1.1.1.431 N309A the 30fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose is lost completely in the mutant. Replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8–9 kJ/mol Yamadazyma tenuis
1.1.1.431 N309D the 30fold preference of the wild-type for D-galactose compared with 2-deoxy-D-galactose is lost completely in the mutant. Comparison of the 2.4 A X-ray crystal structure of mutant N309D bound to NAD+ with the previous structure of the wild-type holoenzyme reveals no major structural perturbations. Replacement of Asn309 with alanine or aspartic acid disrupts the function of the original side chain in donating a hydrogen atom for bonding with the substrate C-2(R) hydroxy group, thus causing a loss of transition-state stabilization energy of 8–9 kJ/mol Yamadazyma tenuis
1.1.1.431 W23F mutant catalyses NADH-dependent reduction of xylose with 4% of the wild-type efficiency (kcat/Km), but improves the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 156 Yamadazyma tenuis
1.1.1.431 W23Y mutant catalyses NADH-dependent reduction of xylose with 1% of the wild-type efficiency (kcat/Km), but improves the wild-type selectivity for utilization of ketones, relative to xylose, by factors of 471 Yamadazyma tenuis

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.431 NADH
-
 Yamadazyma tenuis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.431 2.3
-
 NADPH pH 7.0, 25°C, mutant enzyme D50A Yamadazyma tenuis
1.1.1.431 3.2
-
 NADPH pH 7.0, 25°C, wild-type enzyme Yamadazyma tenuis
1.1.1.431 38
-
 NADH pH 7.0, 25°C, wild-type enzyme Yamadazyma tenuis
1.1.1.431 40
-
 NADH pH 7.0, 25°C, mutant enzyme D50A Yamadazyma tenuis
1.1.1.431 334
-
 xylitol pH 7.0, 25°C, wild-type enzyme Yamadazyma tenuis
1.1.1.431 537
-
 xylitol pH 7.0, 25°C, mutant enzyme D50A Yamadazyma tenuis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.431 D-xylose + NADPH + H+ Yamadazyma tenuis
-
 xylitol + NADP+
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.431 Yamadazyma tenuis O74237
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.431
-
 Yamadazyma tenuis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.431 2-deoxy-D-galactose + NADH
-
 Yamadazyma tenuis ?
-
 ?
1.1.1.431 D-galactose + NADH
-
 Yamadazyma tenuis ?
-
 ?
1.1.1.431 D-glucose + NADH
-
 Yamadazyma tenuis ?
-
 ?
1.1.1.431 D-glyceraldehyde + NADH
-
 Yamadazyma tenuis ?
-
 ?
1.1.1.431 D-xylose + NADH + H+ kcat of wilde-type enzyme increases by a factor of 1.73 when NADPH replaces NADH Yamadazyma tenuis xylitol + NAD+
-
 r
1.1.1.431 D-xylose + NADPH + H+
-
 Yamadazyma tenuis xylitol + NADP+
-
 ?
1.1.1.431 D-xylose + NADPH + H+ kcat of wild-type enzyme increases by a factor of 1.73 when NADPH replaces NADH Yamadazyma tenuis xylitol + NADP+
-
 r
1.1.1.431 additional information in vitro the enzyme also catalyzes the reduction of ketones Yamadazyma tenuis ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.1.1.431 CtXR
-
 Yamadazyma tenuis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.431 25
-
 assay at Yamadazyma tenuis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.431 7
-
 assay at Yamadazyma tenuis

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.431 NADH kcat of wild-type enzyme increases by a factor of 1.73 when NADPH replaces NADH. kcat for NADPH-dependent reduction of xylose by the mutant D50A is three times that for the corresponding NADH-dependent reaction Yamadazyma tenuis
1.1.1.431 NADPH kcat of wild-type enzyme increases by a factor of 1.73 when NADPH replaces NADH. kcat for NADPH-dependent reduction of xylose by the mutant D50A is three times that for the corresponding NADH-dependent reaction Yamadazyma tenuis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.1.1.431 0.0019
-
 NADH pH 7.0, 25°C Yamadazyma tenuis
1.1.1.431 0.02
-
 NADH pH 7.0, 25°C, mutant enzyme D50A Yamadazyma tenuis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.431 0.3
-
 DL-glyceraldehyde pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 0.3
-
 DL-glyceraldehyde pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309D, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 3.7
-
 2-deoxy-D-galactose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309D, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 5.5
-
 D-galactose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309D, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 6.3
-
 2-deoxy-D-galactose pH 7.0, 25°C, cofactor: NADH, mutant enzyme D50A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 6.5
-
 D-xylose pH 7.0, 25°C, cofactor: NADH, mutant enzyme W23Y, kcat/Km value is corrected for the 0.02% of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 8.3
-
 D-glucose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 8.3
-
 D-glucose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309D, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 8.9
-
 DL-glyceraldehyde pH 7.0, 25°C, cofactor: NADH, wild-type enzyme, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 9
-
 D-galactose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 9
-
 D-xylose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309D, kcat/Km value is corrected for the 0.02% of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 9.3
-
 2-deoxy-D-galactose pH 7.0, 25°C, cofactor: NADH, wild-type enzyme, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 14
-
 D-xylose pH 7.0, 25°C, cofactor: NADH, mutant enzyme D50A, kcat/Km value is corrected for the 0.02% of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 17
-
 2-deoxy-D-galactose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 19
-
 D-xylose pH 7.0, 25°C, cofactor: NADH, mutant enzyme N309A, kcat/Km value is corrected for the 0.02% of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 25
-
 D-xylose pH 7.0, 25°C, cofactor: NADH, mutant enzyme W23F, kcat/Km value is corrected for the 0.02% of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 70
-
 D-galactose pH 7.0, 25°C, cofactor: NADH, mutant enzyme D50A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 188
-
 D-glucose pH 7.0, 25°C, cofactor: NADH, mutant enzyme D50A, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 265
-
 D-galactose pH 7.0, 25°C, cofactor: NADH, wild-type enzyme, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 680
-
 D-xylose pH 7.0, 25°C, cofactor: NADH, wild-type enzyme, kcat/Km value is corrected for the 0.02% of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis
1.1.1.431 833
-
 D-glucose pH 7.0, 25°C, cofactor: NADH, wild-type enzyme, kcat/Km value is corrected for the proportion of open-chain free aldehyde in aqueous solution of xylose Yamadazyma tenuis