BRENDA - Enzyme Database show
show all sequences of 1.1.1.209

Structure of the G225P/G226P mutant of mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) ternary complex: implications for the binding of inhibitor and substrate

Dhagat, U.; Endo, S.; Mamiya, H.; Hara, A.; El-Kabbani, O.; Acta Crystallogr. Sect. D 65, 257-265 (2009)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
recombinant wild-type and mutant enzymes expressed in Escherichia coli JM109
Mus musculus
Crystallization (Commentary)
Crystallization
Organism
double mutant G225P/G226P of AKR1C21 in complex with the coenzyme NADP+ and the inhibitor hexoestrol, by hanging-drop vapour-diffusion method, at 2.1 A resolution. Overall fold of the mutant is similar to that of wild-type, apart from the loop region lining the active-site pocket, in which the two glycines at positions 225 and 226 are mutated to prolines which have a more rigid structure, resulting in a larger substrate-binding pocket. The mutation affects the conformation of the adjoining residues Tyr224 and Trp227, resulting in a further opening up of the active-site pocket while the catalytic residues remain unaffected. The inhibitor molecule is bound to the active site with one hydroxyl group pointing towards the catalytic residues, the bulky phenyl rings and the methyl groups of hexoestrol form van der Waals contacts with Trp227, Tyr224, Tyr55 and the nicotinamide ring of NADP+
Mus musculus
Engineering
Amino acid exchange
Commentary
Organism
G225P/G226P
reduced affinity (Km) for both 3alpha- and 17alpha-hydroxysteroid substrates by up to 160fold, shows high selectivity for 5beta-pregnane-3alpha,20alpha-diol in the oxidation of the 3alpha-hydroxy group and, conversely, broad specificity for both 17alpha- and 17beta-hydroxysteroids
Mus musculus
Inhibitors
Inhibitors
Commentary
Organism
Structure
Hexoestrol
-
Mus musculus
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0002
-
4-androstene-3,17-dione
wild-type
Mus musculus
0.0004
-
5alpha-Androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.0004
-
5beta-androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.0005
-
5alpha-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.0006
-
5alpha-Androstan-3alpha-ol-17-one
wild-type
Mus musculus
0.0006
-
5beta-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.0006
-
Epitestosterone
wild-type
Mus musculus
0.0009
-
4-androstene-3,17-dione
mutant G225P/G226P
Mus musculus
0.0013
-
5beta-androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.0029
-
5beta-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
0.0048
-
5alpha-Androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.0097
-
5alpha-Androstan-3alpha-ol-17-one
mutant G225P/G226P
Mus musculus
0.018
-
Epitestosterone
mutant G225P/G226P
Mus musculus
0.023
-
testosterone
mutant G225P/G226P
Mus musculus
0.027
-
5alpha-androstan-17beta-ol-3-one
mutant G225P/G226P
Mus musculus
0.08
-
5alpha-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Mus musculus
Q91WR5
-
-
Purification (Commentary)
Commentary
Organism
recombinant wild-type and mutant enzymes purified to homogeneity
Mus musculus
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4-androstene-3,17-dione + NADPH
-
695325
Mus musculus
? + NADP+
-
-
-
?
5alpha-androstan-17alpha-ol-3-one + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
5alpha-androstan-17beta-ol-3-one + NADP+
wild-type shows no activity with 5alpha-androstan-17beta-ol-3-one
695325
Mus musculus
5alpha-androstan-3,17-dione + NADPH + H+
-
-
-
?
5alpha-androstan-3alpha-ol-17-one + NADPH
-
695325
Mus musculus
? + NADP+
-
-
-
?
5alpha-pregnane-3alpha,20alpha-diol + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
5beta-androstan-17alpha-ol-3-one + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
5beta-pregnane-3alpha,20alpha-diol + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
epitestosterone
-
695325
Mus musculus
? + NADPH
-
-
-
?
additional information
wild-type shows no activity with testosterone and 5alpha-androstan-17beta-ol-3-one
695325
Mus musculus
?
-
-
-
-
testosterone + NADP+
mutant G225P/G226P, wild-type shows no activity with testosterone
695325
Mus musculus
androst-4-en-3,17-dione + NADPH + H+
-
-
-
?
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.04
-
testosterone
mutant G225P/G226P
Mus musculus
0.055
-
5beta-androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.073
-
5alpha-Androstan-3alpha-ol-17-one
wild-type
Mus musculus
0.105
-
5alpha-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.1367
-
4-androstene-3,17-dione
mutant G225P/G226P
Mus musculus
0.14
-
5beta-androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.145
-
5alpha-Androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.15
-
5alpha-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
0.167
-
4-androstene-3,17-dione
wild-type
Mus musculus
0.167
-
5alpha-Androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.183
-
5beta-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
0.2167
-
5alpha-Androstan-3alpha-ol-17-one
mutant G225P/G226P
Mus musculus
0.2167
-
5beta-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.25
-
Epitestosterone
mutant G225P/G226P
Mus musculus
0.3
-
Epitestosterone
wild-type
Mus musculus
0.383
-
5alpha-androstan-17beta-ol-3-one
mutant G225P/G226P
Mus musculus
Cofactor
Cofactor
Commentary
Organism
Structure
NADP+
-
Mus musculus
IC50 Value
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.00026
-
wild-type
Mus musculus
Hexoestrol
0.0006
-
mutant G225P/G226P
Mus musculus
Hexoestrol
Cloned(Commentary) (protein specific)
Commentary
Organism
recombinant wild-type and mutant enzymes expressed in Escherichia coli JM109
Mus musculus
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NADP+
-
Mus musculus
Crystallization (Commentary) (protein specific)
Crystallization
Organism
double mutant G225P/G226P of AKR1C21 in complex with the coenzyme NADP+ and the inhibitor hexoestrol, by hanging-drop vapour-diffusion method, at 2.1 A resolution. Overall fold of the mutant is similar to that of wild-type, apart from the loop region lining the active-site pocket, in which the two glycines at positions 225 and 226 are mutated to prolines which have a more rigid structure, resulting in a larger substrate-binding pocket. The mutation affects the conformation of the adjoining residues Tyr224 and Trp227, resulting in a further opening up of the active-site pocket while the catalytic residues remain unaffected. The inhibitor molecule is bound to the active site with one hydroxyl group pointing towards the catalytic residues, the bulky phenyl rings and the methyl groups of hexoestrol form van der Waals contacts with Trp227, Tyr224, Tyr55 and the nicotinamide ring of NADP+
Mus musculus
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
G225P/G226P
reduced affinity (Km) for both 3alpha- and 17alpha-hydroxysteroid substrates by up to 160fold, shows high selectivity for 5beta-pregnane-3alpha,20alpha-diol in the oxidation of the 3alpha-hydroxy group and, conversely, broad specificity for both 17alpha- and 17beta-hydroxysteroids
Mus musculus
IC50 Value (protein specific)
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
0.00026
-
wild-type
Mus musculus
Hexoestrol
0.0006
-
mutant G225P/G226P
Mus musculus
Hexoestrol
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
Hexoestrol
-
Mus musculus
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
0.0002
-
4-androstene-3,17-dione
wild-type
Mus musculus
0.0004
-
5alpha-Androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.0004
-
5beta-androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.0005
-
5alpha-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.0006
-
5alpha-Androstan-3alpha-ol-17-one
wild-type
Mus musculus
0.0006
-
5beta-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.0006
-
Epitestosterone
wild-type
Mus musculus
0.0009
-
4-androstene-3,17-dione
mutant G225P/G226P
Mus musculus
0.0013
-
5beta-androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.0029
-
5beta-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
0.0048
-
5alpha-Androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.0097
-
5alpha-Androstan-3alpha-ol-17-one
mutant G225P/G226P
Mus musculus
0.018
-
Epitestosterone
mutant G225P/G226P
Mus musculus
0.023
-
testosterone
mutant G225P/G226P
Mus musculus
0.027
-
5alpha-androstan-17beta-ol-3-one
mutant G225P/G226P
Mus musculus
0.08
-
5alpha-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant wild-type and mutant enzymes purified to homogeneity
Mus musculus
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4-androstene-3,17-dione + NADPH
-
695325
Mus musculus
? + NADP+
-
-
-
?
5alpha-androstan-17alpha-ol-3-one + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
5alpha-androstan-17beta-ol-3-one + NADP+
wild-type shows no activity with 5alpha-androstan-17beta-ol-3-one
695325
Mus musculus
5alpha-androstan-3,17-dione + NADPH + H+
-
-
-
?
5alpha-androstan-3alpha-ol-17-one + NADPH
-
695325
Mus musculus
? + NADP+
-
-
-
?
5alpha-pregnane-3alpha,20alpha-diol + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
5beta-androstan-17alpha-ol-3-one + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
5beta-pregnane-3alpha,20alpha-diol + NADP+
-
695325
Mus musculus
? + NADPH
-
-
-
?
epitestosterone
-
695325
Mus musculus
? + NADPH
-
-
-
?
additional information
wild-type shows no activity with testosterone and 5alpha-androstan-17beta-ol-3-one
695325
Mus musculus
?
-
-
-
-
testosterone + NADP+
mutant G225P/G226P, wild-type shows no activity with testosterone
695325
Mus musculus
androst-4-en-3,17-dione + NADPH + H+
-
-
-
?
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
0.04
-
testosterone
mutant G225P/G226P
Mus musculus
0.055
-
5beta-androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.073
-
5alpha-Androstan-3alpha-ol-17-one
wild-type
Mus musculus
0.105
-
5alpha-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.1367
-
4-androstene-3,17-dione
mutant G225P/G226P
Mus musculus
0.14
-
5beta-androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.145
-
5alpha-Androstan-17alpha-ol-3-one
mutant G225P/G226P
Mus musculus
0.15
-
5alpha-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
0.167
-
4-androstene-3,17-dione
wild-type
Mus musculus
0.167
-
5alpha-Androstan-17alpha-ol-3-one
wild-type
Mus musculus
0.183
-
5beta-pregnane-3alpha,20alpha-diol
mutant G225P/G226P
Mus musculus
0.2167
-
5alpha-Androstan-3alpha-ol-17-one
mutant G225P/G226P
Mus musculus
0.2167
-
5beta-pregnane-3alpha,20alpha-diol
wild-type
Mus musculus
0.25
-
Epitestosterone
mutant G225P/G226P
Mus musculus
0.3
-
Epitestosterone
wild-type
Mus musculus
0.383
-
5alpha-androstan-17beta-ol-3-one
mutant G225P/G226P
Mus musculus
Other publictions for EC 1.1.1.209
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
710716
Dhagat
Studies on a Tyr residue criti ...
Mus musculus
Acta Crystallogr. Sect. D
66
198-204
2010
-
-
-
1
3
-
-
36
-
-
-
-
-
2
-
-
-
-
-
-
-
-
12
-
-
-
-
38
-
-
-
-
-
-
-
-
-
-
-
1
3
-
-
-
-
36
-
-
-
-
-
-
-
-
-
-
-
-
12
-
-
-
-
38
-
-
-
-
-
-
-
-
36
36
695325
Dhagat
Structure of the G225P/G226P m ...
Mus musculus
Acta Crystallogr. Sect. D
65
257-265
2009
-
-
1
1
1
-
1
16
-
-
-
-
-
2
-
-
1
-
-
-
-
-
10
-
-
-
-
16
-
-
-
1
-
-
2
-
-
1
1
1
1
-
2
1
-
16
-
-
-
-
-
-
-
1
-
-
-
-
10
-
-
-
-
16
-
-
-
-
-
-
-
-
-
-
685538
Dhagat
Inhibition of 3(17)alpha-hydro ...
Mus musculus
Bioorg. Med. Chem.
16
3245-3254
2008
-
-
-
-
-
-
10
-
-
-
-
1
-
2
-
-
-
-
-
-
-
-
1
-
1
-
-
-
1
-
-
-
-
-
9
-
-
-
-
-
-
-
9
10
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
684185
Dhagat
Structure of 3(17)alpha-hydrox ...
Mus musculus
Acta Crystallogr. Sect. F
63
825-830
2007
-
-
-
1
-
-
-
-
-
-
-
3
-
2
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
667072
El-Kabbani
Crystallization and preliminar ...
Mus musculus
Acta crystallogr. Sect. F
61
688-690
2005
-
-
1
1
-
-
-
-
-
-
1
-
-
2
-
-
1
-
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
389547
Lin
Expression and characterizatio ...
Homo sapiens
Mol. Endocrinol.
11
1971-1984
1997
-
-
1
-
-
-
-
4
-
-
-
-
-
1
-
-
-
-
-
5
3
-
3
-
-
-
-
4
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
5
3
-
3
-
-
-
-
4
-
-
-
-
-
-
-
-
-
-
286234
Nakagawa
Identification of two dihydrod ...
Mus musculus
J. Biochem.
106
633-638
1989
-
-
-
-
-
-
10
13
-
-
2
-
-
3
-
-
1
-
-
1
1
-
27
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10
-
13
-
-
2
-
-
-
-
1
-
1
1
-
27
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
286232
Lau
A 3(17) alpha-hydroxysteroid d ...
Oryctolagus cuniculus
J. Biol. Chem.
257
9444-9449
1982
-
-
-
-
-
-
-
-
1
-
2
-
-
1
-
-
1
-
-
1
-
1
5
1
-
-
-
-
-
-
-
4
-
-
-
-
-
-
4
-
-
-
-
-
-
-
1
-
2
-
-
-
-
1
-
1
-
1
5
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
286233
Lau
Comparison of the multiple for ...
Oryctolagus cuniculus
J. Biol. Chem.
257
9450-9456
1982
-
-
-
-
-
-
1
6
1
-
-
-
-
1
-
-
-
-
-
2
-
-
6
-
-
-
-
-
1
-
-
4
-
-
-
-
-
-
4
-
-
-
-
1
-
6
1
-
-
-
-
-
-
-
-
2
-
-
6
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-