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Literature summary extracted from

  • Shiota, A.; Inaba, S.; Oda, M.
    Effects of active site residues of 3alpha-hydroxysteroid dehydrogenase from Pseudomonas sp. b-0831 on its catalysis and cofactor binding (2018), Biosci. Biotechnol. Biochem., 82, 1702-1707 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.50 overoverexpression with an N-terminal His-tag in Escherichia coli Pseudomonas sp. B-0831
1.1.1.357 recombinant expression of N-terminally His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3). The N-terminal His-tag has little effect on the enzyme function Pseudomonas sp. B-0831

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.50 K157A the NADH binding affinity of K157A mutant is much lower than that of the wild-type, mainly due to loss of a hydrogen bond Pseudomonas sp. B-0831
1.1.1.50 S114A mutant shows higher Km and lower kcat values in both oxidation and reduction reactions Pseudomonas sp. B-0831
1.1.1.50 S114A/Y153F double mutation results in a significant decrease in kcat relative to the single mutant Y153F Pseudomonas sp. B-0831
1.1.1.50 Y153F mutant shows higher Km and lower kcat values in both oxidation and reduction reactions. Loss of hydrogen bonding with NADH upon the Y153F mutation results in increased enthalpy change Pseudomonas sp. B-0831
1.1.1.357 K157A site-directed mutagenesis of a catalytic site residue, the NADH binding affinity of K157A mutant is much lower than that of the wild-type resulting in a decreased kcat Pseudomonas sp. B-0831
1.1.1.357 S114A site-directed mutagenesis of a catalytic site residue, the mutant shows higher Km and lower kcat values in both oxidation and reduction reactions compared to wild-type Pseudomonas sp. B-0831
1.1.1.357 S114A/Y153A site-directed mutagenesis of a catalytic site residue, the double mutant shows higher Km and a highly reduced kcat values in both oxidation and reduction reactions compared to wild-type Pseudomonas sp. B-0831
1.1.1.357 Y153A site-directed mutagenesis of a catalytic site residue, the mutant shows higher Km and lower kcat values in both oxidation and reduction reactions compared to wild-type Pseudomonas sp. B-0831

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.50 51
-
 dehydrocholate 37°C, pH 8.0, wild-type enzyme Pseudomonas sp. B-0831
1.1.1.50 56
-
 cholate 37°C, pH 8.0, wild-type enzyme Pseudomonas sp. B-0831
1.1.1.50 78
-
 cholate 37°C, pH 8.0, mutant enzyme S114A/Y153F Pseudomonas sp. B-0831
1.1.1.50 96
-
 cholate 37°C, pH 8.0, mutant enzyme K157A Pseudomonas sp. B-0831
1.1.1.50 239
-
 dehydrocholate 37°C, pH 8.0, mutant enzyme S114A Pseudomonas sp. B-0831
1.1.1.50 468
-
 cholate 37°C, pH 8.0, mutant enzyme Y153F Pseudomonas sp. B-0831
1.1.1.50 586
-
 cholate 37°C, pH 8.0, mutant enzyme S114A Pseudomonas sp. B-0831
1.1.1.50 2489
-
 dehydrocholate 37°C, pH 8.0, mutant enzyme Y153F Pseudomonas sp. B-0831
1.1.1.357 additional information
-
 additional information steady-state kinetics Pseudomonas sp. B-0831
1.1.1.357 51
-
 Dehydrocholic acid recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 56
-
 cholic acid recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 78
-
 cholic acid recombinant His-tagged mutant S114A/Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 96
-
 cholic acid recombinant His-tagged mutant K157A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 239
-
 Dehydrocholic acid recombinant His-tagged mutant S114A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 468
-
 cholic acid recombinant His-tagged mutant Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 586
-
 cholic acid recombinant His-tagged mutant S114A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 2489
-
 Dehydrocholic acid recombinant His-tagged mutant Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.357 cholic acid + NAD+ Pseudomonas sp. B-0831
-
 dehydrocholic acid + NADH + H+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.50 Pseudomonas sp. B-0831 Q59718
-
-
1.1.1.357 Pseudomonas sp. B-0831
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.50
-
 Pseudomonas sp. B-0831
1.1.1.357 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and dialysis Pseudomonas sp. B-0831

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.50 cholate + NAD+
-
 Pseudomonas sp. B-0831 7alpha,12alpha-dihydroxy-5-beta-cholan-3-one-24-oic acid + NADH + H+
-
 ?
1.1.1.50 dehydrocholate + NADH + H+
-
 Pseudomonas sp. B-0831 ?
-
 ?
1.1.1.357 cholic acid + NAD+
-
 Pseudomonas sp. B-0831 dehydrocholic acid + NADH + H+
-
 r
1.1.1.357 dehydrocholic acid + NADH + H+
-
 Pseudomonas sp. B-0831 cholic acid + NAD+
-
 r
1.1.1.357 additional information NADH binding to the recombinant enzyme Ps3alphaHSD and its mutants is thermodynamically analyzed using isothermal titration calorimetry Pseudomonas sp. B-0831 ?
-
-

Synonyms

EC Number Synonyms Comment Organism
1.1.1.50 3alpha-hydroxysteroid dehydrogenase
-
 Pseudomonas sp. B-0831
1.1.1.50 Ps3alphaHSD
-
 Pseudomonas sp. B-0831
1.1.1.357 3alpha-hydroxysteroid dehydrogenase
-
 Pseudomonas sp. B-0831
1.1.1.357 Ps3alphaHSD
-
 Pseudomonas sp. B-0831

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.357 37
-
 assay at, both reaction directions Pseudomonas sp. B-0831

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.50 0.045
-
 cholate 37°C, pH 8.0, mutant enzyme K157A Pseudomonas sp. B-0831
1.1.1.50 0.12
-
 cholate 37°C, pH 8.0, mutant enzyme S114A/Y153F Pseudomonas sp. B-0831
1.1.1.50 0.62
-
 cholate 37°C, pH 8.0, mutant enzyme Y153F Pseudomonas sp. B-0831
1.1.1.50 0.8
-
 cholate 37°C, pH 8.0, mutant enzyme S114A Pseudomonas sp. B-0831
1.1.1.50 0.89
-
 dehydrocholate 37°C, pH 8.0, mutant enzyme S114A Pseudomonas sp. B-0831
1.1.1.50 1.1
-
 dehydrocholate 37°C, pH 8.0, mutant enzyme Y153F Pseudomonas sp. B-0831
1.1.1.50 23
-
 cholate 37°C, pH 8.0, wild-type enzyme Pseudomonas sp. B-0831
1.1.1.50 48
-
 dehydrocholate 37°C, pH 8.0, wild-type enzyme Pseudomonas sp. B-0831
1.1.1.357 0.012
-
 cholic acid recombinant His-tagged mutant S114A/Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.045
-
 cholic acid recombinant His-tagged mutant K157A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.62
-
 cholic acid recombinant His-tagged mutant Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.8
-
 cholic acid recombinant His-tagged mutant S114A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.89
-
 Dehydrocholic acid recombinant His-tagged mutant S114A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 1.1
-
 Dehydrocholic acid recombinant His-tagged mutant Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 23
-
 cholic acid recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 48
-
 Dehydrocholic acid recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Pseudomonas sp. B-0831

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.357 8
-
 assay at, both reaction directions Pseudomonas sp. B-0831

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.357 NAD+
-
 Pseudomonas sp. B-0831
1.1.1.357 NADH
-
 Pseudomonas sp. B-0831

General Information

EC Number General Information Comment Organism
1.1.1.357 malfunction loss of hydrogen bonding with NADH upon the Y153F mutation results in increased enthalpy change, partially compensated by increased entropy change. The NADH binding affinity of K157A mutant is much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity results in decreased kcat. Compared to the wild-type, the mutants S114A and Y153F show higher Km and lower kcat values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F results in a significant decrease in kcat relative to the single mutant Pseudomonas sp. B-0831
1.1.1.357 additional information residues Ser114, Tyr153, and Lys157 form the catalytic triad. Tyr153 is a catalytic base and Ser114 is a substitute Pseudomonas sp. B-0831

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.357 0.00015
-
 cholic acid recombinant His-tagged mutant S114A/Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.00044
-
 Dehydrocholic acid recombinant His-tagged mutant Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.0005
-
 cholic acid recombinant His-tagged mutant K157A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.0013
-
 cholic acid recombinant His-tagged mutant Y153F, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.0014
-
 cholic acid recombinant His-tagged mutant S114A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.0037
-
 Dehydrocholic acid recombinant His-tagged mutant S114A, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.411
-
 cholic acid recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Pseudomonas sp. B-0831
1.1.1.357 0.94
-
 Dehydrocholic acid recombinant His-tagged wild-type enzyme, pH 8.0, 37°C Pseudomonas sp. B-0831