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Literature summary for 1.3.1.3 extracted from

  • Bauer, P.; Rudolph, K.; Mueller-Uri, F.; Kreis, W.
    Vein patterning 1-encoded progesterone 5beta-reductase: activity-guided improvement of catalytic efficiency (2012), Phytochemistry, 77, 53-59.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
DNA and amino acid sequence determination and analysis, sequence comparisons, expression of wild-type and mutant enzymes in Escherichia coli strain M15 Digitalis lanata
DNA and amino acid sequence determination and analysis, sequence comparisons, expression of wild-type and mutant enzymes in Escherichia coli strain M15 Arabidopsis thaliana

Crystallization (Commentary)

Crystallization (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain M15 by nickel affinity chromatography Digitalis lanata

Protein Variants

Protein Variants Comment Organism
C352G site-directed mutagenesis, mutation at the substrate binding site, the mutant shows reduced activity compared to the wild-type enzyme Digitalis lanata
D181T/L182Q site-directed mutagenesis, mutation at motif V, the mutant shows reduced activity compared to the wild-type enzyme Digitalis lanata
F353M site-directed mutagenesis, mutation at the substrate binding site, the mutant shows similar activity as the wild-type enzyme Digitalis lanata
F353P site-directed mutagenesis, mutation at the substrate binding site, the mutant shows similar activity as the wild-type enzyme Digitalis lanata
G204N site-directed mutagenesis, mutation at motif IV, the mutant shows reduced activity compared to the wild-type enzyme Digitalis lanata
L182Q site-directed mutagenesis, mutation at motif V, the mutant shows similar activity as the wild-type enzyme Digitalis lanata
M150L site-directed mutagenesis, mutation at motif IV, the mutant shows similar activity as the wild-type enzyme Digitalis lanata
N205A site-directed mutagenesis, mutation at the substrate binding site, the mutant shows increased activity compared to the wild-type enzyme Digitalis lanata
N205M site-directed mutagenesis, mutation at the substrate binding site, the mutant shows increased activity compared to the wild-type enzyme Digitalis lanata
N205M/Y156V site-directed mutagenesis, double mutation at the substrate binding site, the mutant shows increased activity compared to the wild-type enzyme Digitalis lanata
R146T site-directed mutagenesis, mutation at motif IV, the mutant shows similar activity as the wild-type enzyme Digitalis lanata
S248M site-directed mutagenesis, mutation near the substrate binding site, inactive mutant Digitalis lanata
T65P site-directed mutagenesis, mutation at near motif II, the mutant shows similar activity as the wild-type enzyme Digitalis lanata
Y156V site-directed mutagenesis, mutation at the substrate binding site, the mutant shows increased activity compared to the wild-type enzyme Digitalis lanata
Y302F site-directed mutagenesis, mutation at motif IV, the mutant shows similar activity as the wild-type enzyme Digitalis lanata

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.063
-
progesterone pH 8.0, 40°C, recombinant mutant N205M Digitalis lanata
0.072
-
progesterone pH 8.0, 40°C, recombinant mutant Y156V Digitalis lanata
0.085
-
progesterone pH 8.0, 40°C, recombinant mutant N205A Digitalis lanata
0.09
-
progesterone pH 8.0, 40°C, recombinant mutant Y156V/N205M Digitalis lanata
0.116
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant wild-type enzyme Arabidopsis thaliana
0.175
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant N205A Digitalis lanata
0.196
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant N205M Digitalis lanata
0.268
-
progesterone pH 8.0, 40°C, recombinant wild-type enzyme Arabidopsis thaliana
0.333
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant wild-type enzyme Digitalis lanata
0.334
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant Y156V/N205M Digitalis lanata
0.362
-
progesterone pH 8.0, 40°C, recombinant wild-type enzyme Digitalis lanata
1.15
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant Y156V Digitalis lanata

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-cyclohexen-1-one + NADPH + H+ Digitalis lanata
-
cyclohexanone + NADP+
-
?
2-cyclohexen-1-one + NADPH + H+ Arabidopsis thaliana
-
cyclohexanone + NADP+
-
?
progesterone + NADPH + H+ Digitalis lanata
-
5beta-pregnane-3,20-dione + NADP+
-
?
progesterone + NADPH + H+ Arabidopsis thaliana
-
5beta-pregnane-3,20-dione + NADP+
-
?

Organism

Organism UniProt Comment Textmining
Arabidopsis thaliana Q9STX2
-
-
Digitalis lanata
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain M15 by nickel affinity chromatography Arabidopsis thaliana

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-cyclohexen-1-one + NADPH + H+
-
Digitalis lanata cyclohexanone + NADP+
-
?
2-cyclohexen-1-one + NADPH + H+
-
Arabidopsis thaliana cyclohexanone + NADP+
-
?
progesterone + NADPH + H+
-
Digitalis lanata 5beta-pregnane-3,20-dione + NADP+
-
?
progesterone + NADPH + H+
-
Arabidopsis thaliana 5beta-pregnane-3,20-dione + NADP+
-
?

Synonyms

Synonyms Comment Organism
P5betaR
-
Digitalis lanata
P5betaR
-
Arabidopsis thaliana
progesterone 5beta-reductase
-
Digitalis lanata
progesterone 5beta-reductase
-
Arabidopsis thaliana

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
assay at Digitalis lanata
40
-
assay at Arabidopsis thaliana

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0075
-
progesterone pH 8.0, 40°C, recombinant mutant Y156V Digitalis lanata
0.011
-
progesterone pH 8.0, 40°C, recombinant mutant Y156V/N205M Digitalis lanata
0.019
-
progesterone pH 8.0, 40°C, recombinant wild-type enzyme Digitalis lanata
0.03
-
progesterone pH 8.0, 40°C, recombinant mutant N205M Digitalis lanata
0.037
-
progesterone pH 8.0, 40°C, recombinant mutant N205A Digitalis lanata
0.17
-
progesterone pH 8.0, 40°C, recombinant wild-type enzyme Arabidopsis thaliana
0.4
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant Y156V/N205M Digitalis lanata
0.475
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant N205M Digitalis lanata
0.49
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant wild-type enzyme Digitalis lanata
0.68
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant Y156V Digitalis lanata
1.1
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant N205A Digitalis lanata
1.11
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant wild-type enzyme Arabidopsis thaliana

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
assay at Digitalis lanata
8
-
assay at Arabidopsis thaliana

Cofactor

Cofactor Comment Organism Structure
NADPH
-
Digitalis lanata
NADPH
-
Arabidopsis thaliana

General Information

General Information Comment Organism
evolution the enzyme belongs to the aldo–keto reductase (AKR) family, sequence comparisons, overview Digitalis lanata
evolution the enzyme belongs to the aldo–keto reductase (AKR) family, sequence comparisons, overview Arabidopsis thaliana
additional information residues Tyr156, Asp205, and Ser248 are responsible for the low catalytic efficiency of the enzyme, substrate binding pocket structure, overview Digitalis lanata
additional information the enzyme shows high catalytic efficiency Arabidopsis thaliana

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.052
-
progesterone pH 8.0, 40°C, recombinant wild-type enzyme Digitalis lanata
0.105
-
progesterone pH 8.0, 40°C, recombinant mutant Y156V Digitalis lanata
0.122
-
progesterone pH 8.0, 40°C, recombinant mutant Y156V/N205M Digitalis lanata
0.432
-
progesterone pH 8.0, 40°C, recombinant mutant N205A Digitalis lanata
0.473
-
progesterone pH 8.0, 40°C, recombinant mutant N205M Digitalis lanata
0.588
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant Y156V Digitalis lanata
0.628
-
progesterone pH 8.0, 40°C, recombinant wild-type enzyme Arabidopsis thaliana
1.19
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant Y156V/N205M Digitalis lanata
1.464
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant wild-type enzyme Digitalis lanata
2.425
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant N205M Digitalis lanata
6.266
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant mutant N205A Digitalis lanata
9.607
-
2-cyclohexen-1-one pH 8.0, 40°C, recombinant wild-type enzyme Arabidopsis thaliana