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Literature summary extracted from

  • Zhang, B.; Zhu, D.; Hu, X.; Zhou, M.; Shang, P.; Lin, S.
    Human 3-alpha hydroxysteroid dehydrogenase type 3 (3alpha-HSD3): The V54L mutation restricting the steroid alternative binding and enhancing the 20alpha-HSD activity (2014), J. Steroid Biochem. Mol. Biol., 141, 135-143.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.50 overexpression of GST-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS Homo sapiens
1.1.1.53 expressed in Escherichia coli BL21(DE3)pLysS cells Homo sapiens

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
1.1.1.50 3alpha-HSD3-NADP+-progesterone complex and 3alpha-HSD3 mutant V54L-NADP+-progesterone complex, sitting drop vapor diffusion method, room temperature, the reservoir solution contains 100 mM sodium cacodylate, pH 6.0, 200 mM ammonium sulfate, 24-26% w/v PEG 3350, with 0.8 mM progesterone, 3-14 days, X-ray diffraction structure determination and analysis at 2.0-2.2 A resolution, molecular replacement with search model, PDB ID 1J96. Progesterone adopts two different binding modes to form complexes within the wild-type enzyme, with one binding mode similar to the orientation of a bile acid (ursodeoxycholate) in the reported ternary complex of human 3alpha-HSD3-NADP+-ursodeoxycholate and the other binding mode resembling the orientation of 20alpha-OHProg in the ternary complex of human 20alpha-HSD-NADP+-20alpha-OHProg Homo sapiens
1.1.1.53 in complex with NADP+ and progesterone, sitting drop vapor diffusion method, using 100 mM sodiumcacodylate buffer (pH 6.0), 200 mM ammonium sulfate, 24-26% (w/v) polyethylene glycol 3350 Homo sapiens

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.50 V54L site-directed mutagenesis, the change renders the sequence identical to that of human 20-alpha hydroxysteroid dehydrogenase. The V54L mutation directly restricts the steroid binding modes to a unique one, which resembles the orientation of 20alpha-OHProg within human 20alpha-HSD. The kinetic study shows that the V54L mutation significantly decreases the 3alpha-HSD activity for the reduction of 5alpha-dihydrotestosterone, while this mutation enhances the 20alpha-HSD activity to convert progesterone Homo sapiens
1.1.1.53 V54L the mutation restricts the steroid alternative binding and enhances the 20alpha-hydroxysteroid dehydrogenase activity of the enzyme Homo sapiens

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.50 0.00084
-
 pregn-4-en-3,20-dione pH 7.4, 37°C, recombinant wild-type enzyme Homo sapiens
1.1.1.50 0.00213
-
 pregn-4-en-3,20-dione pH 7.4, 37°C, recombinant mutant V54L Homo sapiens
1.1.1.50 0.00218
-
 5alpha-dihydrotestosterone pH 7.4, 37°C, recombinant wild-type enzyme Homo sapiens
1.1.1.50 0.07103
-
 5alpha-dihydrotestosterone pH 7.4, 37°C, recombinant mutant V54L Homo sapiens
1.1.1.53 0.00084
-
 progesterone wild type enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 0.00213
-
 progesterone mutant enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 0.00218
-
 5alpha-dihydrotestosterone wild type enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 0.07103
-
 5alpha-dihydrotestosterone mutant enzyme, at pH 7.4 and 37°C Homo sapiens

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.50 5alpha-dihydrotestosterone + NADPH + H+ Homo sapiens
-
 5alpha-androstane-3alpha,17beta-diol + NADP+
-
 r

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.50 Homo sapiens Q04828
-
-
1.1.1.53 Homo sapiens
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.1.1.50 recombinant GST-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS by glutathione affinity chromatography, cleavage of the tag by thrombin, and anion exchange chromatography Homo sapiens
1.1.1.53 glutathione agarose column chromatography and Q-Sepharose column chromatography Homo sapiens

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.50 5alpha-dihydrotestosterone + NADPH + H+
-
 Homo sapiens 5alpha-androstane-3alpha,17beta-diol + NADP+
-
 r
1.1.1.50 pregn-4-en-3,20-dione + NADPH + H+
-
 Homo sapiens 3alpha-hydroxypregn-4-en-20-one + NADP+
-
 r
1.1.1.53 5alpha-dihydrotestosterone + NADP+
-
 Homo sapiens 5alpha-androstane-3,17-dione + NADPH + H+
-
 ?
1.1.1.53 progesterone + NADP+
-
 Homo sapiens 20beta-hydroxy-pregn-4-ene-3-one + NADPH + H+
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.1.1.50 3-alpha hydroxysteroid dehydrogenase type 3
-
 Homo sapiens
1.1.1.50 3alpha-HSD3
-
 Homo sapiens
1.1.1.50 AKR1C1
-
 Homo sapiens
1.1.1.53 3-alpha hydroxysteroid dehydrogenase type 3
-
 Homo sapiens
1.1.1.53 3alpha-HSD3
-
 Homo sapiens

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.50 37
-
 assay at Homo sapiens

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.50 0.0035
-
 pregn-4-en-3,20-dione pH 7.4, 37°C, recombinant wild-type enzyme Homo sapiens
1.1.1.50 0.0392
-
 pregn-4-en-3,20-dione pH 7.4, 37°C, recombinant mutant V54L Homo sapiens
1.1.1.50 0.0612
-
 5alpha-dihydrotestosterone pH 7.4, 37°C, recombinant wild-type enzyme Homo sapiens
1.1.1.50 0.1153
-
 5alpha-dihydrotestosterone pH 7.4, 37°C, recombinant mutant V54L Homo sapiens
1.1.1.53 0.004
-
 progesterone wild type enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 0.039
-
 progesterone mutant enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 0.061
-
 5alpha-dihydrotestosterone wild type enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 0.115
-
 5alpha-dihydrotestosterone mutant enzyme, at pH 7.4 and 37°C Homo sapiens

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.50 7.4
-
 assay at Homo sapiens

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.50 NADP+ binding structure and mechanism, overview Homo sapiens
1.1.1.50 NADPH
-
 Homo sapiens
1.1.1.53 NADP+
-
 Homo sapiens

General Information

EC Number General Information Comment Organism
1.1.1.50 evolution human 3alpha-HSD3 shares 97.8% sequence identity with human 20-alpha hydroxysteroid dehydrogenase (20alpha-HSD) and there is only one amino acid difference (residue 54) that is located in their steroid binding pockets. 20alpha-HSD displays a distinctive ability in transforming progesterone to 20alpha-hydroxy-progesterone Homo sapiens
1.1.1.50 malfunction the V54L mutation significantly decreases the 3alpha-HSD activity for the reduction of 5alpha-dihydrotestosterone, while this mutation enhances the 20alpha-HSD activity to convert progesterone Homo sapiens
1.1.1.50 physiological function 3-alpha hydroxysteroid dehydrogenase type 3 has an essential role in the inactivation of 5alpha-dihydrotestosterone preventing binding and activation of androgen receptor from overflowing androgen Homo sapiens

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.50 1.623
-
 5alpha-dihydrotestosterone pH 7.4, 37°C, recombinant mutant V54L Homo sapiens
1.1.1.50 4.167
-
 pregn-4-en-3,20-dione pH 7.4, 37°C, recombinant wild-type enzyme Homo sapiens
1.1.1.50 18.4
-
 pregn-4-en-3,20-dione pH 7.4, 37°C, recombinant mutant V54L Homo sapiens
1.1.1.50 28.07
-
 5alpha-dihydrotestosterone pH 7.4, 37°C, recombinant wild-type enzyme Homo sapiens
1.1.1.53 100
-
 5alpha-dihydrotestosterone mutant enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 250
-
 progesterone wild type enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 1100
-
 progesterone mutant enzyme, at pH 7.4 and 37°C Homo sapiens
1.1.1.53 1680
-
 5alpha-dihydrotestosterone wild type enzyme, at pH 7.4 and 37°C Homo sapiens