Information on EC 1.1.1.213 - 3alpha-hydroxysteroid 3-dehydrogenase (Re-specific) and Organism(s) Rattus norvegicus and UniProt Accession P23457

enzyme is stereoselective for benzo[g]chrysene-11S,12S-dihydrodiol

-

?

17beta-hydroxy-5alpha-androstan-3-one + NAD(P)H + H+

3alpha,17beta-dihydroxy-5alpha-androstan + NAD(P)+

-

-

?

2 tibolone + 2 NADPH + 2 H+

3alpha-hydroxytibolone + 3beta-hydroxytibolone + 2 NADP+

5alpha-androstan-3,17-dione + NAD(P)H

3alpha-hydroxy-5alpha-androstan-17-one + NAD(P)+

-

-

r

benzo[c]phenanthrene-3,4-dihydrodiol + NADP+

? + NADPH + H+

enzyme metabolizes both stereoisomers of benzo[c]phenanthrene-3,4-dihydrodiol

-

?

(2Z)-2-(5-hydroxy-4,6-dimethyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N,N-di(prop-2-en-1-yl)hydrazinecarbothioamide + NADPH + H+

?

-

-

r

(5beta,20R)-20-hydroxypregnan-3-one + NADH + H+

?

-

-

r

1-(4'nitrophenyl)prop-2-en-1-ol + NAD+

1-(4'nitrophenyl)prop-2-en-1-one + NADH

-

-

?

1-(4'nitrophenyl)prop-2-yn-1-ol + NAD+

1-(4'nitrophenyl)prop-2-yn-1-one + NADH

-

-

?

1-acenaphthenol + NAD(P)+

1-acenaphthenone + NAD(P)H + H+

-

pH 9.0

-

?

1-acetophenone + NADPH

1-phenylethanol + NADP+

-

-

?

17beta-hydroxy-5alpha-androstan-3-one + NAD(P)H

5alpha-androstane-3alpha,17beta-diol + NAD(P)+

-

the rate of dihydrotestosterone reduction over back-conversion is 2.4 and 5.9 for prostate and epididymis, respectively

347987

-

347987

r

17beta-hydroxy-5alpha-androstan-3-one + NAD(P)H + H+

3alpha,17beta-dihydroxy-5alpha-androstan + NAD(P)+

17beta-hydroxy-5alpha-androstan-3-one + NADPH

3alpha,17beta-dihydroxy-5alpha-androstan + NADP+

-

347979

-

347979

r

2-acetylpyridine + NADPH

1-(2-pyridyl)ethanol + NADP+

-

stereoselective catalysis producing mainly the (S)-alcohols

-

?

2-decalone + NADH

? + NAD+

-

pH 6.0

-

?

3-acetylpyridine + NADPH

1-(3-pyridyl)ethanol + NADP+

-

low activity, stereoselective catalysis producing mainly the (S)-alcohols

-

?

3alpha,5alpha-allopregnenolone + NAD(P)+

5alpha-dihydroprogesterone + NAD(P)H

3alpha-androstanediol + NAD+

5alpha-dihydrotestosterone + NADH + H+

-

steroid reduction direction is preferred

-

r

3alpha-hydroxy-5alpha-androstan-17-one + NAD+

5alpha-androstan-3,17-dione + NADH

4'-methoxyacetophenone + NADPH + H+

1-(4-methoxyphenyl)ethanol + NADP+

-

R,S-enantiomeric product

-

?

4-acetylpyridine + NADPH + H+

(S)-1-(4-pyridyl)ethanol + NADP+

-

-

?

4-acetylpyridine + NADPH + H+

1-(4-pyridyl)ethanol + NADP+

-

stereoselective catalysis producing mainly the (S)-alcohols

-

?

4-androsten-3alpha-ol-17-one + NADH + H+

4-androsten-3alpha,17beta-diol + NAD+

-

AKR1C17

-

r

4-androstene-3,17-dione + NADH + H+

4-androsten-3alpha-ol-17-one + NAD+

-

low activity

-

r

4-bromoacetophenone + NADPH + H+

1-(4-bromophenyl)ethanol + NADP+

-

-

?

4-cyanoacetophenone + NADPH + H+

1-(4-cyanophenyl)ethanol + NADP+

-

-

?

4-methylacetophenone + NADPH + H+

1-(4-methylphenyl)ethanol + NADP+

-

R,S-enantiomeric product

-

?

4-nitroacetophenone + NAD(P)H

1-(4-nitrophenyl)ethanol + NAD(P)+

-

pH 6.0

-

?

4-nitroacetophenone + NADPH + H+

1-(4-nitrophenyl)ethanol + NADP+

-

-

?

4-nitrobenzaldehyde + NAD(P)H

(4-nitrophenyl)methanol + NAD(P)+

5alpha-androstan-3,17-dione + NAD(P)H

3alpha-hydroxy-5alpha-androstan-17-one + NAD(P)+

-

pH 6.0 for reduction, pH 7.0 and 9.0 for androsterone oxidation

-

r

5alpha-androstan-3alpha-ol-17-one + NADH + H+

5beta-androstan-3alpha,17beta-diol + NAD+

-

-

r

5alpha-androstane-3,17-dione + NADH

5alpha-androstan-3alpha-ol-17-one + NAD+

-

low activity

AKR1C9

-

r

5alpha-dihydrotestosterone + NADH

?

-

low activity

-

?

5alpha-dihydrotestosterone + NADPH

5alpha-androstane-3alpha,17beta-diol + NADP+

5alpha-dihydrotestosterone + NADPH + H+

3alpha-androstanediol + NADP+

5alpha-pregnan-3,20-dione + NAD(P)H + H+

5alpha-pregnan-3alpha-ol-20-one + NAD(P)+

-

regulation of the amount of allosteric agonists that can bind to the GABA receptor in brain

-

r

5alpha-pregnane-20alpha-ol-3-one + NADH

5alpha-pregnane-3,20alpha-diol + NAD+

-

-

?

5alpha-pregnane-3,20-dione + NADH

5alpha-pregnan-3alpha-ol-20-one + NAD+

-

low activity

-

r

5beta-androstan-3alpha-ol-17-one + NAD(P)+

5beta-androstane-3,17-dione + NAD(P)H

-

-

r

5beta-androstan-3alpha-ol-17-one + NADH

5beta-androstane-3alpha,17beta-diol + NAD+

-

-

r

5beta-androstane-3,17-dione + NADH

5beta-androstan-3alpha-ol-17-one + NAD+

-

-

r

5beta-pregnan-3alpha-ol-20-one + NAD(P)+

5beta-pregnane-3,20-dione + NAD(P)H

-

-

r

5beta-pregnane-17alpha,20beta,21-triol-3,11-dione + NAD(P)H

5beta-pregnane-3alpha,17alpha,20beta,21-tetraol-11-one + NAD(P)+

5beta-pregnane-20alpha-ol-3-one + NADH + H+

5beta-pregnane-3,20alpha-diol + NAD+

-

-

?

5beta-pregnane-3,20-dione + NADH + H+

5beta-pregnan-3alpha-ol-20-one + NAD+

-

-

r

6-tert-butyl-2,3-epoxy-4-hydroxy-5-cyclohexen-1-one + NADH

?

-

the enzyme produces both R- and S-form products in a ratio of 2.3 : 1

-

?

7alpha,12alpha-dihydroxy-5beta-cholestan-3-one + NADPH + H+

5beta-cholestane-3alpha,7alpha,12alpha-triol + NADP+

-

722157

-

?

7alpha-hydroxy-5beta-cholestan-3-one + NADPH + H+

5beta-cholestane-3alpha,7alpha-diol + NADP+

-

722157

-

?

9,10-phenanthrenequinone + NAD(P)H

? + NAD(P)+

9,10-phenanthrenequinone + NADH

?

-

-

?

acetophenone + NADPH + H+

1-phenylethanol + NADP+

-

-

?

androstan-17beta-ol-3-one + NADH

androstan-3alpha,17beta-diol + NAD+

-

-

?

androstan-3,17-dione + NADH

androstan-3alpha-ol-17-one + NAD+

-

-

?

androsterone + NAD(P)+

5alpha-androstane-3,17-dione + NAD(P)H

-

-

r

androsterone + NADP+

5alpha-androstane-3,17-dione + NADPH

-

-

r

benzenedihydrodiol + NAD(P)+

?

-

pH 9.0

-

?

beta-muricholic acid + NADH

?

-

low activity

-

?

chenodeoxycholic acid + NADH

?

-

-

?

cholic acid + NADH

?

-

low activity

-

?

dehydrolithocholic acid + NADH + H+

?

-

-

?

deoxycholic acid + NADH

?

-

low activity

-

?

diacetyl + NADH

?

-

-

?

etiocholane-3,17-dione + NADH

etiocholan-3alpha-ol-17-one + NAD+

-

-

?

glycochenodeoxycholic acid + NADH

?

-

-

?

glycolithocholic acid + NADH

?

-

-

?

glycoursodeoxycholic acid + NADH

?

-

-

?

hyodeoxycholic acid + NADH

?

-

low activity

-

?

isatin + NADH

?

-

-

?

lithocholic acid + NADH

?

-

-

?

murocholic acid + NADH

?

-

low activity

-

?

pregnane-11beta,17alpha,21-triol-3,20-dione + NADH

pregnane-3alpha,11beta,17alpha,21-tetrol-20-one + NAD+

-

-

?

progesterone + NAD(P)H

3alpha-hydroxy-pregn-4-ene-20-one + NAD(P)+

-

-

r

prostaglandin + NADP+

? + NADPH

-

types A1, A2, B1, B2, D2, E1, E2, F1, F2alpha and 15-keto-prostaglandinE2 and F2alpha

-

?

prostaglandin-F2alpha + NADP+

prostaglandin F2 + prostaglandin B2 + NADPH

-

-

?

taurolithocholic acid + NADH

?

-

-

?

tauroursodeoxycholic acid + NADH

?

-

-

?

testosterone + NAD(P)H + H+

4-androsten-3alpha,17beta-diol + NAD(P)+

-

-

r

testosterone + NADH

?

-

low activity

-

?

ursodeoxycholic acid + NADH

?

-

-

?

additional information

?

-

11 entries

17beta-hydroxy-5alpha-androstan-3-one + NAD(P)H + H+

3alpha,17beta-dihydroxy-5alpha-androstan + NAD(P)+

-

-

?

2 tibolone + 2 NADPH + 2 H+

3alpha-hydroxytibolone + 3beta-hydroxytibolone + 2 NADP+

i.e. tibolone, a 3-ketosteroid androgen receptor, conversion to potent estrogen receptor alpha agonists, tibolone induces estrogen receptor alpha-dependent gene promoter activity through cis-acting estrogen response elements, increases the stimulatory effect of TGF-beta on Smad-dependent gene promoter activity, and enhances prostaglandin E2-induced activity of transcription factor Runx2, overview

3alpha-hydroxytibolone is the primary metabolite

-

r

17beta-hydroxy-5alpha-androstan-3-one + NAD(P)H + H+

3alpha,17beta-dihydroxy-5alpha-androstan + NAD(P)+

3alpha,5alpha-allopregnenolone + NAD(P)+

5alpha-dihydroprogesterone + NAD(P)H

-

i.e. 3alpha,5alpha-tetrahydroprogesterone or 3alpha,5alpha-THP, the enzyme catalyzes the biosynthesis and oxidation of 3alpha,5alpha-reduced neurosteroids as allopregnanolone, which stimulates GABAA receptors, sciatic nerves-induced analgesia results in increased enzyme levels in neuropathic rats, overview

-

r

3alpha-androstanediol + NAD+

5alpha-dihydrotestosterone + NADH + H+

-

steroid reduction direction is preferred

-

r

4-acetylpyridine + NADPH + H+

(S)-1-(4-pyridyl)ethanol + NADP+

-

-

?

5alpha-dihydrotestosterone + NADPH

5alpha-androstane-3alpha,17beta-diol + NADP+

-

-

?

5alpha-dihydrotestosterone + NADPH + H+

3alpha-androstanediol + NADP+

-

steroid reduction direction is preferred

-

r

5alpha-pregnan-3,20-dione + NAD(P)H + H+

5alpha-pregnan-3alpha-ol-20-one + NAD(P)+

-

regulation of the amount of allosteric agonists that can bind to the GABA receptor in brain

-

r

5beta-pregnane-17alpha,20beta,21-triol-3,11-dione + NAD(P)H

5beta-pregnane-3alpha,17alpha,20beta,21-tetraol-11-one + NAD(P)+

-

hepatic reduction in animal tissues

-

?

acetophenone + NADPH + H+

1-phenylethanol + NADP+

-

-

?

androsterone + NADP+

5alpha-androstane-3,17-dione + NADPH

-

-

r

additional information

?

-

NAD+

-

NADH

-

NADP+

-

347990, 687639, 697310

NADPH

-

NAD(P)+

NAD(P)H

NAD+

NADH

NADP+

NADPH

additional information

6 entries

-

additional information

-

non-metallo enzyme

DMSO

33% inhibition

naphthalene-1,2-dione

naphthalene-1,2-dione leads to the time and concentration dependent irreversible inactivation of AKR1C9 via alkylation

(E/Z)-sulfindac

-

wild-type, W86Y and W227Y

1,10-phenanthroline

-

wild-type, W86Y and W227Y

1,7-phenanthroline

-

wild-type, W86Y and W227Y

1-(4'nitrophenyl)prop-2-en-1-ol

-

inactivation dependent on NAD+ concentration, optimal at 0.5-1.0 mM NAD+, 2-mercaptoethanol prvides a concentration-dependent protection

1-(4'nitrophenyl)prop-2-en-1-one

-

inactivation can be retarded markedly in a concentration-dependent manner by both NADH and NADPH. Competitive inhibitor of NAD+ binding, measured for androsterone oxidation

1-(4'nitrophenyl)prop-2-yn-1-one

-

competitive inhibitor of NAD+ binding, measured for androsterone oxidation

17beta-bromoacetoxy-5alpha-dihydrotestosterone

-

inactivation by modification of steroid-binding site

4-androstene-3,17-dione

-

versus substrate, the products, 4-androstene-3,17-dione and NADH, inhibit the activity uncompetitively and competitively, respectively, with respect to NAD+ in the presence of a saturated concentration of 0.004 mM of the substrate

6alpha-Methylprednisolone

-

acetaminophen

-

non-competitive, only androsterone oxidation, pH 7.0

acetylenic ketones

-

inactivation by forming Michael adducts with enzyme nucleophiles

-

arachidonic acid

-

347980, 347982

aspirin

-

salicylate, non-competitive, only androsterone oxidation, pH 7.0

Betamethasone

-

non-competitive

cacodylate

-

citrate

-

cortisol

-

competitive

cortisone

-

competitive

Cu2+

-

100% inhibition at 0.1 mM

D-glucose 6-phosphate

-

dexamethasone

-

non-competitive

Flufenamic acid

-

wild-type, W86Y and W227Y

Hexestrol

-

Ibuprofen

-

competitive

indomethacin

iodoacetate

-

50% inhibition at 0.1 mM

Ketamine

-

specific inhibitor for AKR1C17, but no inhibition of AKR1C9

Meclofenamic acid

Mefenamic acid

-

wild-type, W86Y and W227Y

NADH

-

the products, 4-androstene-3,17-dione and NADH, inhibit the activity uncompetitively and competitively, respectively, with respect to NAD+ in the presence of a saturated concentration of 0.004 mM of the substrate

non-steroidal anti-inflamatory drug

-

Oxyphenybutazone

-

competitive

p-chloromercuribenzoate

-

non-competitive with respect to dihydrocortisone 46-100% inhibition at 0.01 mM, preincubation with NADH lowers the inhibitory effect

ponalrestat

-

wild-type, W86Y and W227Y, weak inhibitor

Prednisolone

-

competitive

Prednisone

-

competitive

progesterone

-

competitive to testosterone

Prostaglandin

prostaglandin A1

-

prostaglandin A2alpha

-

Prostaglandin B1

-

prostaglandin E1

-

prostaglandin F1alpha

-

pyrazole

-

10% at 0.4 mM

salicylate

-

non-competitive

testosterone

Tolmetin

-

competitive, only androsterone oxidation, pH 7.0

Zomepirac

-

competitive, only androsterone oxidation, pH 7.0

zopolrestat

additional information

-

not inhibited by dicoumarol, disulfiram and barbiturates. Inhibitory potency of non-steroidal anti-inflamatory drugs and salicylates falls sharply as the pH is increased from 6 to 9

-

additional information

prostaglandin E2 increases AKR1C9 gene promoter activity and mRNA expression, AKR1C9 promoter activity is also enhanced by overexpression of protein kinase A catalytic subunit or transcription factor C/EBPdelta, whicb is reduced by dominant negative C/EBPdelta competition or C/EBPdelta antisense expression, overview

-

0.0013 - 0.0441

3alpha-hydroxy-5alpha-androstan-17-one

0.0011 - 0.0304

5alpha-androstan-3,17-dione

0.004 - 0.821

NAD+

-

0.0057 - 0.0312

NADH

-

0.0052 - 0.1515

NADP+

-

0.0022 - 0.0081

NADPH

-

0.0027

(5beta,20R)-20-hydroxypregnan-3-one

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.0012 - 0.726

17beta-hydroxy-5alpha-androstan-3-one

6 entries

3.54 - 33.7

2-decalone

-

0.029 - 0.408

3alpha-hydroxy-5alpha-androstan-17-one

0.163 - 0.211

4-nitrobenzaldehyde

-

0.0017 - 0.0042

5alpha-androstan-3,17-dione

0.00065

5alpha-androstane-3,17-dione

-

pH 7.0, 25, reduction reaction

0.781 - 7.331

5alpha-androstane-3alpha,17beta-diol

0.0021

5beta-androstane-3,17-dione

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.01

5beta-pregnane-17alpha,20beta,21-triol-3,11-dione

-

0.0024

5beta-pregnane-3,20-dione

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.023

6-tert-butyl-2,3-epoxy-4-hydroxy-5-cyclohexen-1-one

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.00122 - 0.038

9,10-phenanthrenequinone

0.0041

androsterone

-

pH 7.0, 25, oxidation reaction

0.004

Dehydrolithocholic acid

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.42

diacetyl

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.022

isatin

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.04 - 0.94

NAD+

0.015 - 0.033

NADH

0.04

NADP+

-

0.0001

Prostaglandin

-

additional information

(11S,12S)-11,12-dihydrobenzo[g]chrysene-11,12-diol

8 entries

0.00063 - 0.85

(2Z)-2-(5-hydroxy-4,6-dimethyl-2-oxo-1,2-dihydro-3H-indol-3-ylidene)-N,N-di(prop-2-en-1-yl)hydrazinecarbothioamide

0.115

(5beta,20R)-20-hydroxypregnan-3-one

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

1.13 - 1.25

3alpha-hydroxy-5alpha-androstan-17-one

0.267 - 0.283

5alpha-androstan-3,17-dione

0.42

5alpha-androstane-3,17-dione

-

pH 7.0, 25, reduction reaction

0.037 - 0.43

5alpha-dihydroprogesterone

0.047

5beta-androstane-3,17-dione

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.105

5beta-pregnane-3,20-dione

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.1

6-tert-butyl-2,3-epoxy-4-hydroxy-5-cyclohexen-1-one

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.183 - 3.93

9,10-phenanthrenequinone

0.0053 - 0.83

androsterone

5 entries

0.022

Dehydrolithocholic acid

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.167

diacetyl

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

0.283

isatin

-

pH 7.4, 25°C, recombinant wild-type AKR1C17

1.13 - 1.43

NAD+

0.283 - 0.367

NADH

additional information

-

0.0022 - 0.018

4-androstene-3,17-dione

0.0075

6alpha-Methylprednisolone

-

androsterone oxidation

0.006

arachidonic acid

0.65

aspirin

-

for androsterone oxidation

0.0045

Betamethasone

-

androsterone oxidation

0.19

cortisol

-

androsterone oxidation

0.285

cortisone

-

androsterone oxidation

0.01

dexamethasone

-

androsterone oxidation

0.0002 - 0.001

indomethacin

0.017

NADH

-

0.0175

Prednisolone

-

androsterone oxidation

0.0175

Prednisone

-

androsterone oxidation

0.0031

prostaglandin A1

-

androsterone oxidation

0.003

prostaglandin A2alpha

-

0.0008

Prostaglandin B1

-

androsterone oxidation

0.0075

prostaglandin E1

-

androsterone oxidation

0.012

prostaglandin F1alpha

-

androsterone oxidation

0.115 - 0.75

salicylate

-

additional information

-

more Ki values given for different inhibitors of androsterone oxidation, 9,10-phenanthrenequinone- and 5alpha-androstane-3,17-dione reduction

-

0.000735 - 0.00333

indomethacin

0.08

Q190A mutant, androsterone oxidation with NAD+

0.1

S166A mutant, androsterone oxidation with NAD+

0.81

Y216S mutant, androsterone oxidation with NADP+

1.55

recombinant wild type enzyme, androsterone oxidation with NAD+

1.6

wild type homogeneous recombinant AKR1C9, at 25°C

0.0005 - 0.0053

-

for prostaglandins A1, A2, B1, B2, D2, E1, E2, F1, F2alpha at concentrations of 0.06 mM

0.0007 - 0.0012

-

for 15-keto-prostaglandins E2 and F2alpha at concentrations of 0.06 mM

0.006

-

purified recombinant mutant W227A, substrate androsterone

0.008 - 4

-

determined for several substrates,with best activities for 9,10-phenanthrenequinone, 4-nitrobezaldehyde and androsterone

0.01

-

purified recombinant mutant F118A, substrate androsterone

0.03

-

purified recombinant mutant F129A, substrate androsterone

0.059

-

W86Y mutant

0.07

-

recombinant AKR1C17 in cell extract, substrate 4-androsten-3alpha-ol-17-one

0.08

0.09

-

purified recombinant mutant N306A, substrate androsterone

0.1

-

purified recombinant mutant Y310A, substrate androsterone

0.23

-

purified recombinant AKR1C17, substrate 4-androsten-3alpha-ol-17-one

0.674

-

W148Y mutant

0.86

-

purified recombinant mutant T24A, substrate androsterone

0.96

-

purified recombinant mutant T226A, substrate androsterone

1.5

-

native enzyme

1.6

-

purified recombinant wild-type enzyme, substrate androsterone

1.93

-

2

-

for androsterone

3.19

-

9540

-

purified enzyme

additional information

9

assay at

7

-

assay at

654726, 654756, 687635

7.4

-

assay at

673101, 685529

7.4 - 9.5

-

recombinant enzyme

5.5 - 11

-

25

assay at

37

assay at

25

-

assay at

347979, 347980, 347982, 347985, 654726, 684676, 687635

37

-

assay at

347987, 673101, 685529

-

347990, 687639, 697310

UniProt

-

-

fetal

-

-

-

lumbar, of naive, sham-operated and neuropathic rats, enzyme expression analysis and cellular distribution in neurons and glial cells, immunohistochemic analysis, overview

spinal cord

-

abundant enzyme expression

-

additional information

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additional information

P23457 pBLAST

DIDH_RAT

322

0

37028

Swiss-Prot

Show Sequence

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1lwi, download, 3D-view

37000

1 * 37000, SDS-PAGE

33000

33500

-

1 * 33500, SDS-PAGE

34000

34600

-

gel filtration

36000

-

recombinant AKR1C17, gel filtration

37000

37029

-

1 * 37029

37030

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predicted molecular weight from the cDNA

347981

74000 - 75000

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native PAGE, kidney enzyme

monomer

1 * 37000, SDS-PAGE

?

monomer

additional information

vapor diffusion techniques using ammonium sulfate as precipitant

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F118A

largest changes in kcat/Km

F129A

largest changes in kcat/Km, F129A alters the stereochemical preference from oxidizing predominantly the S,S-stereoisomer to oxidizing predominantly the R,R-stereoisomer of benzo[g]chrysene-11,12-dihydrodiol

L54A

mutant shows intermediate changes in kcat/Km versus wild type

N167A

site-directed mutagenesis, most impaired enzyme

N306A

mutant shows intermediate changes in kcat/Km versus wild type

Q190A

site-directed mutagenesis, decreased binding affinity to NADP(H), only binding of cofactor is affected, residue is located at the catalytic cente

R276M

elimination of salt bridge between Arg276 and the 2'-phosphate of AMP results in 100fold decrease of the affinity to NADP(H)

S166A

site-directed mutagenesis, decreased binding affinity to NADP(H), only binding of cofactor is affected, residue is located at the catalytic center

T226A

smallest change in kcat/Km is observed when alanine is used to substitute hydrophilic residues

T24A

smallest change in kcat/Km is observed when alanine is used to substitute hydrophilic residues

W227A

largest changes in kcat/Km

Y216S

site-directed mutagenesis, decreased binding affinity to NADP(H), only binding of cofactor is affected, residue is located at the catalytic cente

Y310A

mutant shows intermediate changes in kcat/Km versus wild type

C217A

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resistant to inactivation by secosteroids, therefore Cys217 is the point of covalent attachment of acetylenic ketones

D50E

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1/30th catalytic efficiency of wild type, unlikely to be the general amino acid for catalysis

D50N

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1/30th catalytic efficiency of wild type, unlikely to be the general amino acid for catalysis

E276R

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site-directed mutagenesis, the mutation alters the cofactor specificity of AKR1C17 from NAD+ to NADP+, the switch is analogy th the residues of AKRc9 and its cofactor specificity, overview

F118A

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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

F129A

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site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

H117A

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1/500th catalytic efficiency of wild type, unlikely to be the general amino acid for catalysis

K84M

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inactive, unable to bind steroids

K84R

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inactive, unable to bind steroids

L54A

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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

N306A

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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

Q270K

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site-directed mutagenesis, the mutation alters the cofactor specificity of AKR1C17 from NAD+ to NADP+, the switch is analogy th the residues of AKRc9 and its cofactor specificity, overview

Q270K/E276R

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site-directed mutagenesis, the mutation alters the cofactor specificity of AKR1C17 from NAD+ to NADP+, the switch is analogy th the residues of AKRc9 and its cofactor specificity, overview

R276E

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site-directed mutagenesis, the mutant shows increased preference for the oxidation reaction compared to the wild-type enzyme

R276G

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site-directed mutagenesis, the mutant shows slightly increased preference for the reduction reaction compared to the wild-type enzyme

R276M

T226A

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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

T24A

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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

W148Y

W227A

W227Y

W86Y

Y205F

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kinetically indistinguishable from the wild type, no general amino acid for catalysis in 3alpha-hydroxysteroid dehydrogenase

Y310A

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site-directed mutagenesis, reduced activity compared to the wild-type enzyme, effects of mutation on binding constants and kinetics, overview

Y55F

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inactive, unable to perform steroid oxidoreduction, strongest candidate for the general amino acid

Y55S

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inactive, strongest candidate for the general amino acid

additional information

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positions of Tyr/Lys pair are conserved across the aldo-keto reductase and short-chain dehydrogenase/reductase family. Tyr retains ability to form ternary complex and acts as general acid

-80°C

-10°C, 3 weeks, activity declines about 50%

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-10°C, most purified preparation, 5 days, loses between 80% and 100% of activity

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-70°C, 20 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM beta-mercaptoethanol, 30% glycerol

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-80°C, 20 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM DTT, 30% glycerol

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347982, 347985

3°C, first ammonium sulfate fraction, stable at least 3 weeks

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3°C, most purified preparation, 5 days, loses 50% of activity

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anion exchange and Blue Sepharose column

N167A chromatofocusing and gel filtration introduced between the to others chromatography steps

purification of recombinant wild type and mutants of AKR1C9

ammonium sulfate precipitation, Ca2(PO4)2 gel extraction, ethanol precipitation

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anion exchange and Blue Sepharose column

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CaCl2 and ammonium sulfate fractionation, DEAE-cellulose- and hydroxyapatite chromatography, chromatofocusing, Sephadex G-100

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347979, 347980, 347982, 347985

native enzyme 73.3fold from liver by ammonium sulfate, anion-exchange chromatography, gel filtration, and affinity chromatography

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recombinant wild-type and mutant AKR1C17s from Escherichia coli strain BL21(DE3)

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recombinant wild-type and mutant enzymes from Escherichia coli C41(DE3), to homogeneity

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expression and promoter activity analysis, genetic enzyme regulation, overview

expression of recombinant wild type and mutants of AKR1C9

expression of wild-type and mutant AKR1C17s in Escherichia coli strain BL21(DE3)

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full-length cDNA gene is expressed in Escherichia coli DH5alpha

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overexpression of AKR1C9 in Escherichia coli

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688137

overexpression of wild-type and mutant enzymes in Escherichia coli C41(DE3)

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plasmid DNA containing cDNA is transformed in Escherichia coli HB101, cDNA is used for sequencing

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347981

stable expression of wild-type and mutant enzymes in HEK-293 cells

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wild-type enzyme and mutants are overexpressed in Escherichia coli DH5alpha

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drug development

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the enzyme is a target for treatment of chronic pain in neuropathic diseases, overview

347979

Askonas, L.J.; Ricigliano, J.W.; Penning, T.M.

The kinetic mechanism catalysed by homogeneous rat liver 3alpha-hydroxysteroid dehydrogenase. Evidence for binary and ternary dead-end complexes containing non-steroidal anti-inflammatory drugs

Biochem. J.

278

835-841

1991

Rattus norvegicus

1898369

Penning, T.M.; Sharp, R.B

Prostaglandin dehydrogenase activity of purified rat liver 3alpha-hydroxysteroid dehydrogenase

Biochem. Biophys. Res. Commun.

148

646-652

1987

Rattus norvegicus

3479982

347981

Pawlowski, J.E.; Huizinga, M.; Penning, T.M.

Cloning and sequencing of the cDNA for rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase

J. Biol. Chem.

266

8820-8825

1991

Rattus norvegicus, Rattus norvegicus Sprangue-Dawley

1840601

Penning, T.M.; Mukharji, I.; Barrows, S.; Talalay, P.

Purification and properties of a 3alpha-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs

Biochem. J.

222

601-611

1984

Rattus norvegicus, Rattus norvegicus Sprangue-Dawley

6435601

Tomkins, G.M.

A mammalian 3alpha-hydroxysteroid dehydrogenase

J. Biol. Chem.

218

437-447

1956

Rattus norvegicus

13278351

Ricigliano, J.W.; Penning, T.M.

Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3alpha-hydroxysteroid dehydrogenase

Biochem. J.

269

749-755

1990

Rattus norvegicus, Rattus norvegicus Sprangue-Dawley

2390066

347987

Span, P.N.; Sweep, C.G.J.; Benraad, T.J.; Smals, A.G.H.

3alpha-Hydroxysteroid oxidoreductase activities in dihydrotestosterone degradation and back-formation in rat prostate and epididymis

J. Steroid Biochem. Mol. Biol.

58

319-324

1996

Rattus norvegicus

8836171

Jez, J.M.; Schlegel, B.P.; Penning, T.M.

Characterization of the substrate binding site in rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase. The roles of tryptophans in ligand binding and protein fluorescence

J. Biol. Chem.

271

30190-30198

1996

Rattus norvegicus

8939970

Penning, T.M.; Bennett, M.J.; Smith-Hoog, S.; Schlegel, B.P.; Jez, J.M.; Lewis, M.

Structure and function of 3alpha-hydroxysteroid dehydrogenase

Steroids

62

101-111

1997

Rattus norvegicus

9029723

Ma, H.; Ratnam, K.; Penning, T.M.

Mutation of nicotinamide pocket residues in rat liver 3alpha-hydroxysteroid dehydrogenase reveals different modes of cofactor binding

Biochemistry

39

102-109

2000

Rattus norvegicus (P23457)

10625484

Heredia, V.V.; Penning, T.M.

Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis

Biochemistry

43

12028-12037

2004

Rattus norvegicus

15379543

Heredia, V.V.; Cooper, W.C.; Kruger, R.G.; Jin, Y.; Penning, T.M.

Alanine scanning mutagenesis of the testosterone binding site of rat 3alpha-hydroxysteroid dehydrogenase demonstrates contact residues influence the rate-determining step

Biochemistry

43

5832-5841

2004

Rattus norvegicus

15134457

Uwai, K.; Konno, N.; Kitamura, S.; Ohta, S.; Takeshita, M.

Purification and characterization of rat liver enzyme catalyzing stereoselective reduction of acetylpyridines

Chirality

17

494-500

2005

Rattus norvegicus

16113996

Papari-Zareei, M.; Brandmaier, A.; Auchus, R.J.

Arginine 276 controls the directional preference of AKR1C9 (rat liver 3alpha-hydroxysteroid dehydrogenase) in human embryonic kidney 293 cells

Endocrinology

147

1591-1597

2006

Rattus norvegicus

16357042

Sanai, M.; Endo, S.; Matsunaga, T.; Ishikura, S.; Tajima, K.; El-Kabbani, O.; Hara, A.

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol

Arch. Biochem. Biophys.

464

122-129

2007

Rattus norvegicus

17475203

Uwai, K.; Konno, N.; Yasuta, Y.; Takeshita, M.

Electronic effects of para-substitution on acetophenones in the reaction of rat liver 3alpha-hydroxysteroid dehydrogenase

Bioorg. Med. Chem.

16

1084-1089

2008

Rattus norvegicus

18006320

Cooper, W.C.; Jin, Y.; Penning, T.M.

Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate: the example of rat liver 3alpha-HSD (AKR1C9)

J. Biol. Chem.

282

33484-33493

2007

Rattus norvegicus

17848571

McCarthy, T.L.; Hochberg, R.B.; Labaree, D.C.; Centrella, M.

3-ketosteroid reductase activity and expression by fetal rat osteoblasts

J. Biol. Chem.

282

34003-34012

2007

Rattus norvegicus (P23457)

17905737

688137

Azzarello, J.; Fung, K.M.; Lin, H.K.

Tissue distribution of human AKR1C3 and rat homolog in adult genitourinary system

J. Histochem. Cytochem.

56

853-861

2008

Homo sapiens, Rattus norvegicus

18574251

Meyer, L.; Venard, C.; Schaeffer, V.; Patte-Mensah, C.; Mensah-Nyagan, A.G.

The biological activity of 3alpha-hydroxysteroid oxido-reductase in the spinal cord regulates thermal and mechanical pain thresholds after sciatic nerve injury

Neurobiol. Dis.

30

30-41

2008

Rattus norvegicus

18291663