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Literature summary extracted from

  • Xu, G.; Zhu, C.; Li, A.; Ni, Y.; Han, R.; Zhou, J.; Ni, Y.
    Engineering an alcohol dehydrogenase for balancing kinetics in NADPH regeneration with 1,4-butanediol as a cosubstrate (2019), ACS Sust. Chem. Eng., 7, 15706-15714 .
No PubMed abstract available

Application

EC Number Application Comment Organism
1.1.1.2 synthesis engineering ADHs for regenerating NADPH by oxidation of diols Vanderwaltozyma polyspora

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.2 recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL(DE3) Vanderwaltozyma polyspora

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.2 F197W site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 additional information engineering an alcohol dehydrogenase for balancing kinetics in NADPH regeneration with 1,4-butanediol as a cosubstrate. Identification of the key residues of KpADH by molecular dynamics simulations Vanderwaltozyma polyspora
1.1.1.2 S196A site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 S196A/F197W site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 V84I site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 V84I/Y127M site-directed mutagenesis, mutant KpADHV84I/Y127M exhibits a lower KM of 15.1 mM and a higher kcat of 30.1/ min than wild-type KpADH. The ratio of kcat/KM toward 2-hydroxytetrahydrofuran (2-HTHF), in comparison to 1,4-BD, in KpADHV84I/Y127M is dramatically reduced by almost 100fold compared to wild-type KpADH, which is advantageous for NADPH regeneration Vanderwaltozyma polyspora
1.1.1.2 V84I/Y12C site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 Y127C site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 Y127C/S196A site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 Y127M site-directed mutagenesis, kinetics compared to the wild-type enzyme Vanderwaltozyma polyspora

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.2 additional information
-
 additional information kinetic analysis of recombinant wild-type an d mutant enzymes, overview Vanderwaltozyma polyspora
1.1.1.2 0.0648
-
 NADP+ pH 9.5, 30°C, recombinant wild-type enzyme, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.0768
-
 NADP+ pH 9.5, 30°C, recombinant mutant V127M, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.0807
-
 NADP+ pH 9.5, 30°C, recombinant mutant V84I/Y127M, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.1
-
 NADP+ pH 9.5, 30°C, recombinant mutant S196A/F197W, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.107
-
 NADP+ pH 9.5, 30°C, recombinant mutant F197W, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.107
-
 NADP+ pH 9.5, 30°C, recombinant mutant V127C, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.159
-
 NADP+ pH 9.5, 30°C, recombinant mutant V84I, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.456
-
 NADP+ pH 9.5, 30°C, recombinant mutant S196A, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.462
-
 NADP+ pH 9.5, 30°C, recombinant mutant V84I/Y127C, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.565
-
 NADP+ pH 9.5, 30°C, recombinant mutant Y127C/S196A, with 2-hydroxytetrahydrofuran Vanderwaltozyma polyspora
1.1.1.2 0.85
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant S196A/F197W Vanderwaltozyma polyspora
1.1.1.2 1.55
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant S196A Vanderwaltozyma polyspora
1.1.1.2 1.59
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant F197W Vanderwaltozyma polyspora
1.1.1.2 1.9
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I Vanderwaltozyma polyspora
1.1.1.2 2.23
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 2.4
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V127C Vanderwaltozyma polyspora
1.1.1.2 4.39
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I/Y127M Vanderwaltozyma polyspora
1.1.1.2 4.7
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I/Y127C Vanderwaltozyma polyspora
1.1.1.2 5.9
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V127M Vanderwaltozyma polyspora
1.1.1.2 6.38
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant Y127C/S196A Vanderwaltozyma polyspora
1.1.1.2 15.1
-
 1,4-Butanediol pH 9.5, 30°C, recombinant mutant V84I/Y127M Vanderwaltozyma polyspora

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.2 Vanderwaltozyma polyspora
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.2 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL(DE3) by nickel affinity chromatography Vanderwaltozyma polyspora

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.2 1,4-butanediol + NADP+
-
 Vanderwaltozyma polyspora 4-hydroxybutanal + NADPH + H+
-
 r
1.1.1.2 1,5-pentanediol + NADP+
-
 Vanderwaltozyma polyspora 5-hydroxypentanal + NADPH + H+
-
 r
1.1.1.2 1,6-hexanediol + NADP+
-
 Vanderwaltozyma polyspora 6-hydroxyhexanal + NADPH + H+
-
 r
1.1.1.2 2-hydroxytetrahydrofuran + NADP+
-
 Vanderwaltozyma polyspora 2-oxotetrahydrofuran + NADPH + H+
-
 ir
1.1.1.2 additional information the oxidation of 1,4-BD to 4-hydroxybutanal is the first, reversible, rate-limiting step in the NADPH regeneration system with 1,4-butanediol (1,4-BD). It is followed by spontaneous formation of 2-hydroxytetrahydrofuran (2-HTHF) from 4-hydroxybutanal via cyclization. The second enzymatic step, the reduction of 2-HTHF into gamma-butyrolactone, is irreversible, liberating 2 equiv. of NAD(P)H Vanderwaltozyma polyspora ?
-
-

Synonyms

EC Number Synonyms Comment Organism
1.1.1.2 KpADH
-
 Vanderwaltozyma polyspora
1.1.1.2 NADP+-dependent alcohol dehydrogenase
-
 Vanderwaltozyma polyspora

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.2 30
-
-
 Vanderwaltozyma polyspora

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.2 0.000038
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant S196A Vanderwaltozyma polyspora
1.1.1.2 0.000053
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I/Y127C Vanderwaltozyma polyspora
1.1.1.2 0.000062
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant Y127C/S196A Vanderwaltozyma polyspora
1.1.1.2 0.000074
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant S196A/F197W Vanderwaltozyma polyspora
1.1.1.2 0.000077
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V127C Vanderwaltozyma polyspora
1.1.1.2 0.00014
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I/Y127M Vanderwaltozyma polyspora
1.1.1.2 0.00018
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant F197W Vanderwaltozyma polyspora
1.1.1.2 0.00022
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I Vanderwaltozyma polyspora
1.1.1.2 0.00039
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V127M Vanderwaltozyma polyspora
1.1.1.2 0.00044
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 0.502
-
 1,4-Butanediol pH 9.5, 30°C, mutant V84I/Y127M Vanderwaltozyma polyspora

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.2 9.5
-
-
 Vanderwaltozyma polyspora

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.2 NADP+
-
 Vanderwaltozyma polyspora
1.1.1.2 NADPH
-
 Vanderwaltozyma polyspora

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.2 0.0000032
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V127C Vanderwaltozyma polyspora
1.1.1.2 0.0000066
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V127M Vanderwaltozyma polyspora
1.1.1.2 0.0000097
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant Y127C/S196A Vanderwaltozyma polyspora
1.1.1.2 0.000011
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I/Y127C Vanderwaltozyma polyspora
1.1.1.2 0.000012
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I Vanderwaltozyma polyspora
1.1.1.2 0.000032
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant V84I/Y127M Vanderwaltozyma polyspora
1.1.1.2 0.000087
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant S196A/F197W Vanderwaltozyma polyspora
1.1.1.2 0.00011
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant F197W Vanderwaltozyma polyspora
1.1.1.2 0.0002
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant wild-type enzyme Vanderwaltozyma polyspora
1.1.1.2 0.00025
-
 2-hydroxytetrahydrofuran pH 9.5, 30°C, recombinant mutant S196A Vanderwaltozyma polyspora
1.1.1.2 0.033
-
 1,4-Butanediol pH 9.5, 30°C, mutant V84I/Y127M Vanderwaltozyma polyspora