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Information on EC 1.1.1.357 - 3alpha-hydroxysteroid 3-dehydrogenase and Organism(s) Comamonas testosteroni and UniProt Accession P80702
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1.1.1.357 3alpha-hydroxysteroid 3-dehydrogenaseIUBMB Comments
The enzyme acts on multiple 3alpha-hydroxysteroids, such as androsterone and 5 alpha-dihydrotestosterone. The mammalian enzymes are involved in inactivation of steroid hormones, while the bacterial enzymes are involved in steroid degradation. This entry stands for enzymes whose stereo-specificity with respect to NAD+ or NADP+ is not known. [cf. EC 1.1.1.50, 3alpha-hydroxysteroid 3-dehydrogenase (Si-specific) and EC 1.1.1.213, 3alpha-hydroxysteroid 3-dehydrogenase (Re-specific)].
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Word Map
- 1.1.1.357
- ileal
-
ibabp
-
farnesoid
-
enterohepatic
- akr1c2
- 3alpha-hydroxysteroids
- glycocholate
- 3alpha-hsds
- gcda
- glycochenodeoxycholate
- drug development
The taxonomic range for the selected organisms is: Comamonas testosteroni
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota
Synonyms
bile acid binding protein, aldo-keto reductase family 1 member c3, akr1c33, 3alpha-hsd type iii, aldo-keto reductase family 1 member c2, type 1 3alpha-hsd, 3alpha-hydroxysteroid dehydrogenase type iii, hsd 28, dihydrodiol dehydrogenase 4, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+
rate-limiting step in the reaction is the release of NADH and proton. P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis, catalytic mechanism, overview
a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+
the reaction catalyzed by the enzyme shows an ordered bi bi kinetic mechanism with NAD+ being the first substrate to be bound and NADH the last product to be released. The rate-limiting steps along the reaction pathway is the release of NADH for reaction with andosterone or androsterol, while the hydride transfer for 2-decalol is rate limiting for the overall reaction. Structure of the transition state by measuring the rate constant of a reaction upon substitution of a heavy atom for a light one at or adjacent to the bond cleavage during the reaction
a 3alpha-hydroxysteroid + NAD(P)+ = a 3-oxosteroid + NAD(P)H + H+
rapid equilibrium ordered bi bi kinetic mechanism
PATHWAY SOURCE
PATHWAYS
-
-, -, -
SYSTEMATIC NAME
IUBMB Comments
3alpha-hydroxysteroid:NAD(P)+ 3-oxidoreductase
The enzyme acts on multiple 3alpha-hydroxysteroids, such as androsterone and 5 alpha-dihydrotestosterone. The mammalian enzymes are involved in inactivation of steroid hormones, while the bacterial enzymes are involved in steroid degradation. This entry stands for enzymes whose stereo-specificity with respect to NAD+ or NADP+ is not known. [cf. EC 1.1.1.50, 3alpha-hydroxysteroid 3-dehydrogenase (Si-specific) and EC 1.1.1.213, 3alpha-hydroxysteroid 3-dehydrogenase (Re-specific)].
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone + NAD(P)H + H+
2-(1H-imidazol-1-yl)-1-(4-methoxyphenyl)-2-methylpropan-1-ol + NAD(P)+
2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone i.e. NKI 42255
-
-
?
3-dehydro-alpha-ecdysone + NAD(P)H + H+
alpha-ecdysone + NAD(P)+
-
-
-
?
3-dehydro-beta-ecdysone + NAD(P)H + H+
beta-ecdysone + NAD(P)+
-
-
-
?
3alpha-hydroxydesogestrel + NAD(P)+
3-oxodesogestrel + NAD(P)H + H+
-
-
-
?
4-nitroacetophenone + NAD(P)H + H+
1-(4-nitrophenyl)ethanol + NAD(P)+
-
-
-
?
4-nitrobenzaldehyde + NAD(P)H + H+
(4-nitrophenyl)methanol + NAD(P)+
-
-
-
?
5alpha-dihydrotestosterone + NAD(P)H + H+
(5alpha,17beta)-androstane-3,17-diol + NAD(P)+
-
-
-
?
5beta-dihydrotestosterone + NAD(P)H + H+
(5beta,17beta)-androstane-3,17-diol + NAD(P)+
-
-
-
?
a 3alpha-hydroxysteroid + NAD(P)+
a 3-oxosteroid + NAD(P)H + H+
-
-
-
?
a 3alpha-hydroxysteroid + NAD+
a 3-oxosteroid + NADH + H+
-
-
-
r
androstandiol + NAD(P)+
(5alpha,17beta)-17-hydroxyandrostan-3-one + NAD(P)H + H+
-
-
-
?
androstandione + NAD(P)H + H+
3-hydroxyandrostan-17-one + NAD(P)+
-
-
-
?
androstenol + NAD+
5alpha-androst-16-en-3-one + NADH + H+
-
-
-
r
androsterone + NAD(P)+
(5alpha)-androstane-3,17-dione + NAD(P)H + H+
-
-
-
?
cholic acid + NAD(P)+
(5beta,7alpha,8xi,12alpha)-7,12-dihydroxy-3-oxocholan-24-oic acid + NAD(P)H + H+
-
-
-
?
deoxycholic acid + NADH
12alpha-hydroxy-3-oxo-5beta-cholan-24-oic acid + NAD+
-
-
-
r
epiandrosterone + NAD(P)+
(5alpha)-androstane-3,17-dione + NAD(P)H + H+
-
-
-
?
fusidic acid + NAD(P)+
(2Z)-2-[(4alpha,5alpha,8alpha,9beta,11alpha,13alpha,14beta,16beta,17Z)-16-(acetyloxy)-11-hydroxy-4,8,10,14-tetramethyl-3-oxogonan-17-ylidene]-6-methylhept-5-enoic acid + NAD(P)H + H+
-
-
-
?
fusidic acid + NADP+
(2Z)-2-[(4alpha,5alpha,8alpha,9beta,11alpha,13alpha,14beta,16beta,17Z)-16-(acetyloxy)-11-hydroxy-4,8,10,14-tetramethyl-3-oxogonan-17-ylidene]-6-methylhept-5-enoic acid + NADPH + H+
-
-
-
?
metyrapol + NAD(P)+
2-methyl-1,2-di(pyridin-3-yl)propan-1-one + NAD(P)H + H+
-
-
-
?
metyrapone + NAD(P)H + H+
2-methyl-1,2-di(pyridin-3-yl)propan-1-ol + NAD(P)+
-
-
-
?
androsterone + NAD+
5alpha-androstan-3,17-dione + NADH + H+
-
-
-
r
androsterone + NAD+
5alpha-androstan-3,17-dione + NADH + H+
rate-limiting step in the reaction is the release of NADH
-
-
r
additional information
?
-
no activity with testosterone, 3-oxo-desogestrel, progesterone, 4-nitrobenzalcohol, trans-benzene dihydrodiol and cis-benzene dihydrodiol
-
-
?
additional information
?
-
-
no activity with testosterone, 3-oxo-desogestrel, progesterone, 4-nitrobenzalcohol, trans-benzene dihydrodiol and cis-benzene dihydrodiol
-
-
?
additional information
?
-
no activity with testosterone and progesterone
-
-
?
additional information
?
-
recombinant 3alpha-HSD/CR protein is active with a variety of C1927 steroid substrates, catalyzing the 3-oxo reduction and 3alpha-hydroxy dehydrogenation of steroids of the androstane and bile acid series with Km-values in the low micromolar range, 1842 microM
-
-
?
additional information
?
-
recombinant enzyme catalyzes carbonyl reduction of nonsteroidal aldehyde and ketone compounds e.g. metyrapone, 4-nitrobenzaldehyde and insecticide NKI 42255, no reverse oxidation of the formed hydroxy derivatives 4-nitrobenzalcohol and NKI 42455 observed
-
-
?
additional information
?
-
the enzyme catalyzes reversibly the oxidoreduction of 3alpha-hydroxyl groups of steroid hormones
-
-
?
additional information
?
-
3alpha-hydroxysteroid dehydrogenase/carbonyl reductase catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with the rate-limiting step being the release of NADH in the overall reaction. No enzyme activity is detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. Role of remote substrate binding interactions contributing to the rate enhancement by 3apha-HSD/CR, overview. Uniform binding improves catalysis with simple cyclic alcohols, differential binding improves catalysis of steroid substrates. Reaction product identifcation by LC-MS/MS analysis
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
a 3alpha-hydroxysteroid + NAD(P)+
a 3-oxosteroid + NAD(P)H + H+
-
-
-
?
a 3alpha-hydroxysteroid + NAD+
a 3-oxosteroid + NADH + H+
-
-
-
r
androsterone + NAD+
5alpha-androstan-3,17-dione + NADH + H+
-
-
-
r
additional information
?
-
the enzyme catalyzes reversibly the oxidoreduction of 3alpha-hydroxyl groups of steroid hormones
-
-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1
2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone
pH 7.0, temperature not specified in the publication, NAD(P)H
0.025
3-dehydro-alpha-ecdysone
pH 7.0, temperature not specified in the publication, NAD(P)H
0.035
3-dehydro-beta-ecdysone
pH 7.0, temperature not specified in the publication, NAD(P)H
0.0152
3alpha-hydroxydesogestrel
pH 8.9, temperature not specified in the publication, NAD(P)+
0.9
4-Nitroacetophenone
pH 7.0, temperature not specified in the publication, NAD(P)H
1.09
4-nitrobenzaldehyde
pH 7.0, temperature not specified in the publication, NAD(P)H
0.0161
5alpha-dihydrotestosterone
pH 7.0, temperature not specified in the publication, NAD(P)H
0.0111
5beta-dihydrotestosterone
pH 7.0, temperature not specified in the publication, NAD(P)H
0.0328
androstandiol
pH 8.9, temperature not specified in the publication, NAD(P)+
0.0357
androstandione
pH 7.0, temperature not specified in the publication, NAD(P)H
0.0301
cholic acid
pH 8.9, temperature not specified in the publication, NAD(P)+
0.0207
epiandrosterone
pH 8.9, temperature not specified in the publication, NAD(P)+
2.1
metyrapol
pH 8.9, temperature not specified in the publication, NAD(P)+
0.61
Metyrapone
pH 7.0, temperature not specified in the publication, NAD(P)H
0.0028
androsterone
reombinant His-tagged enzyme, pH 10.5, 25°C
0.0244
androsterone
pH 8.9, temperature not specified in the publication, NAD(P)+
0.003
Fusidic acid
pH 8.9, temperature not specified in the publication, NAD(P)+
0.0061
Fusidic acid
pH 8.9, temperature not specified in the publication
0.14
NAD+
reombinant His-tagged enzyme, pH 10.5, 25°C, with androstenol
0.39
NAD+
reombinant His-tagged enzyme, pH 10.5, 25°C, with androsterone
additional information
additional information
steady-state kinetics, and stopped-flow study, kinetics of enzyme mutants P185A, P185G, T188A, and T188S showing an increase in kcat, Ka drosterone and KiNAD and equal primary isotope effects of DV and D(V/K)
-
additional information
additional information
steady-state kinetics of 3alpha-HSD/CR catalyzed reaction of NADþ with androsterone and the truncated analogues, overview. The uniform binding energy from the B-ring of steroids with the active site of 3alpha-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in kcat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3alpha-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3a-HSD/CR catalysis, resulting in the increases in kcat and kcat/KB
-
additional information
additional information
steady-state kinetics and thermodynamics. The effects of temperature on kcat/Km for 3alpha-HSD/CR acting on androsterone, 2-decalol, and cyclohexanol show the reactions are entropically favorable but enthalpically unfavorable. Thermodynamic analysis from the temperature-dependent values of Km and kcat shows the binding of the E-NAD+ complex with either 2-decalol or cyclohexanol to form the ternary complex is endothermic and entropy-driven, and the subsequent conversion to the transition state is both enthalpically and entropically unfavorable
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
178571
androsterone
reombinant His-tagged enzyme, pH 10.5, 25°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
15.4
recombinant untagged enzyme purified via deoxycholic acid affinity chromatography, pH 11.0, 25°C
3.94
recombinant His-tagged enzyme purified via Ni2+ affinity chromatography, pH 11.0, 25°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
enzyme 3alpha-HSD/CR belongs to the short chain dehydrogenase/ reductase (SDR) superfamily
malfunction
mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH. Mutants P185A, P185G, T188A, and T188S show an increase the dissociation of the nucleotide cofactor, thereby increasing the rate of release of the product and producing the rate-limiting step in the hydride transfer
metabolism
enzyme is involved in inactivation of steroid hormones
additional information
catalytic tetrad N86-S114-Y155-K159, catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD+ causes the appearance of T188-P191 in the binary complex, functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis, overview. Simulated molecular modeling gives results that are consistent with the conformational change in the substrate-binding loop after NAD+ binding. These results indicate that P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis. Homology structure modeling, overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). DIDH_COMTE
257
0
26392
Swiss-Prot
Secretory Pathway (Reliability: 2)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
26400
26400
calculated from deduced amino acid sequence comprising 258 residues
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
homodimer
non-covalent homodimer, 2 * 23000, about, recombinant enzyme, SDS-PAGE
additional information
secondary structures of the wild-type and mutants of P185A, P185G, T188A and T188S 3alpha-HSD/CRs are assessed by CD spectroscopy by measuring the ellipticity in the 190-250 nm range at room temperature
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
P185A
site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme
P185G
site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme
T188A
site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme
T188S
site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme
W173F/P185W
site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme
W173F/T188W
site-directed mutagenesis, analysis of kinetics and structure compared to the wild-type enzyme
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
in one step using metal chelate chromatography, purity of the protein is more than 98%
recombinant His-tagged and untagged enzyme from Escherchia coli strain BL21(DE3) nickel affinity chromatography, and by deoxycholic acid affinity chromatography, respectively, the latter to 94-98% purity. Deoxycholic acid is linked to the resin Sepharose 4B with the aid of the spacers as cyanuric chloride and ethanediamine. Adsorption analysis by adapting the adsorption isotherm to a specific binding model, the Scatchard analysis model, overview. Comparison of purification methods between synthetic affinity medium and Ni2+-Sepharose column
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3)
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography to homogeneity
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
3alpha-HSD/CR gene cloned into pET15b vector with an N-terminal His tag sequence and transformation of Escherichia coli BL21(DE3) pLysS cells
gene hsdA, recombinant expression of His-tagged and untagged enzyme in Escherchia coli strain BL21(DE3)
recombinant expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
recombinant His-tagged enzyme overexpression in Escherichia coli strain BL21(DE3)
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme is induced in the presence of testosterone and progesterone due to the steroids preventing repressor repA from binding the regulatory regions
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Oppermann, U.C.; Maser, E.
Characterization of a 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase from the gram-negative bacterium Comamonas testosteroni
Eur. J. Biochem.
241
744-749
1996
Comamonas testosteroni (P80702), Comamonas testosteroni, Comamonas testosteroni ATCC 11996 (P80702), Comamonas testosteroni ATCC 11996, Pseudomonas sp. (P80701)
Mobus, E.; Maser, E.
Molecular cloning, overexpression, and characterization of steroid-inducible 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni. A novel member of the short-chain dehydrogenase/reductase superfamily
J. Biol. Chem.
273
30888-30896
1998
Comamonas testosteroni (P80702), Comamonas testosteroni ATCC 11996 (P80702)
Hwang, C.C.; Chang, P.R.; Wang, T.P.
Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase
Chem. Biol. Interact.
276
133-140
2017
Comamonas testosteroni (P80702)
Yang, H.; Fang, Y.; Wang, Z.; Zhang, L.
One step affinity recovery of 3alpha-hydroxysteroid dehydrogenase from cloned Escherichia coli
J. Chromatogr.
991
79-84
2015
Comamonas testosteroni (P80702), Comamonas testosteroni ATCC 11996 (P80702)
Hwang, C.C.; Chang, Y.H.; Lee, H.J.; Wang, T.P.; Su, Y.M.; Chen, H.W.; Liang, P.H.
The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni
PLoS ONE
8
e63594
2013
Comamonas testosteroni (P80702)
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