BRENDA - Enzyme Database show
show all sequences of 1.1.1.248

Molecular modeling and site-directed mutagenesis reveal the benzylisoquinoline binding site of the short-chain dehydrogenase/reductase salutaridine reductase

Geissler, R.; Brandt, W.; Ziegler, J.; Plant Physiol. 143, 1493-1503 (2007)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
salR, DNA and amino acid sequence determination and analysis, overexpression of the His-tagged enzyme in Escherichia coli
Papaver bracteatum
Crystallization (Commentary)
Crystallization
Organism
construction of a homology model of the Papaver bracteatum SalR based on the X-ray structure of human carbonyl reductase 1, overview
Papaver bracteatum
Engineering
Amino acid exchange
Commentary
Organism
F104A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
K240E
site-directed mutagenesis, inactive mutant
Papaver bracteatum
L266A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
L266S
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
L266V
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
M271T
site-directed mutagenesis, inactive mutant
Papaver bracteatum
N152A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
N272T
site-directed mutagenesis, inactive mutant
Papaver bracteatum
R44E
site-directed mutagenesis, the mutant enzyme shows altered cofactor specificity and utilizes also NADH in contrast to the wild-type enzyme
Papaver bracteatum
R48E
site-directed mutagenesis, the mutant enzyme shows altered cofactor specificity and utilizes also NADH in contrast to the wild-type enzyme
Papaver bracteatum
S180A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Papaver bracteatum
V106A
site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme
Papaver bracteatum
Y236F
site-directed mutagenesis, inactive mutant
Papaver bracteatum
Inhibitors
Inhibitors
Commentary
Organism
Structure
salutaridine
-
Papaver bracteatum
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetics of wild-type and mutant enzymes, overview
Papaver bracteatum
0.0015
-
(7S)-salutaridinol
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
0.0035
-
NADPH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
0.007
-
NADP+
pH 6.0, 40C, recombinant enzyme, with salutaridinol
Papaver bracteatum
0.0079
-
salutaridine
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
1.19
-
NADH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
34050
-
1 * 36600, recombinant His-tagged enzyme, SDS-PAGE, 1 * 34050, sequence calculation, the tertiary structure comprises the typical short-chain dehydrogenase/reductase family alpha/beta folding pattern including the four additional helices alphaF-1 to alphaF-4 assumed to prevent the dimerization, modeling and analysis, overview
Papaver bracteatum
36600
-
1 * 36600, recombinant His-tagged enzyme, SDS-PAGE, 1 * 34050, sequence calculation, the tertiary structure comprises the typical short-chain dehydrogenase/reductase family alpha/beta folding pattern including the four additional helices alphaF-1 to alphaF-4 assumed to prevent the dimerization, modeling and analysis, overview
Papaver bracteatum
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
additional information
Papaver bracteatum
the enzyme is involved in the biosynthesis of morphine
?
-
-
-
salutaridine + NADPH + H+
Papaver bracteatum
-
(7S)-salutaridinol + NADP+
-
-
r
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Papaver bracteatum
A4UHT7
-
-
Purification (Commentary)
Commentary
Organism
recombinant His-tagged enzyme from Escherichia coli by cobalt affinity chromatography
Papaver bracteatum
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
additional information
the enzyme is involved in the biosynthesis of morphine
689571
Papaver bracteatum
?
-
-
-
-
additional information
interaction of benzylisoquinoline alkaloids with enzymes, modeling, overview
689571
Papaver bracteatum
?
-
-
-
-
salutaridine + NAD(P)H + H+
NADPH is the highly preferred cofactor, the enzyme shows high substrate specificity, no activity with tropinone, (2)-menthone, codeinone and dehydroreticulinium ion, or nordehydroreticuline. Asp152, Ser180, Tyr236, and Lys240 are involved in the proton transfer system for the reduction of salutaridine, substrate-active site binding structure, overview
689571
Papaver bracteatum
(7S)-salutaridinol + NAD(P)+
the enzyme produces only the (7S)-salutaridinol stereoisomer as product, not the stereoisomer 7-epi-salutaridinol
-
-
r
salutaridine + NADPH + H+
-
689571
Papaver bracteatum
(7S)-salutaridinol + NADP+
-
-
-
r
Subunits
Subunits
Commentary
Organism
monomer
1 * 36600, recombinant His-tagged enzyme, SDS-PAGE, 1 * 34050, sequence calculation, the tertiary structure comprises the typical short-chain dehydrogenase/reductase family alpha/beta folding pattern including the four additional helices alphaF'-1 to alphaF'-4 assumed to prevent the dimerization, modeling and analysis, overview
Papaver bracteatum
Temperature Optimum [C]
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
40
-
broad optimum
Papaver bracteatum
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.1
-
NADPH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
2.3
-
salutaridine
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
3.7
-
NADH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
21.6
-
(7S)-salutaridinol
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
21.9
-
NADP+
pH 6.0, 40C, recombinant enzyme, with salutaridinol
Papaver bracteatum
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
6
-
reduction reaction
Papaver bracteatum
9.5
-
oxidation reaction
Papaver bracteatum
pH Range
pH Minimum
pH Maximum
Commentary
Organism
4.5
7.5
the enzyme shows a steep decrease by 90% of activity within 1.5 pH units on both sides of the optimum at pH 6.0 for the reduction reaction
Papaver bracteatum
Cofactor
Cofactor
Commentary
Organism
Structure
NAD+
-
Papaver bracteatum
NADH
strong preference for NADPH over NADH
Papaver bracteatum
NADP+
-
Papaver bracteatum
NADPH
strong preference for NADPH over NADH
Papaver bracteatum
Ki Value [mM]
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.14
-
salutaridine
pH 6.0, recombinant enzyme
Papaver bracteatum
pI Value
Organism
Commentary
pI Value Maximum
pI Value
Papaver bracteatum
sequence calculation
-
4.72
Cloned(Commentary) (protein specific)
Commentary
Organism
salR, DNA and amino acid sequence determination and analysis, overexpression of the His-tagged enzyme in Escherichia coli
Papaver bracteatum
Cofactor (protein specific)
Cofactor
Commentary
Organism
Structure
NAD+
-
Papaver bracteatum
NADH
strong preference for NADPH over NADH
Papaver bracteatum
NADP+
-
Papaver bracteatum
NADPH
strong preference for NADPH over NADH
Papaver bracteatum
Crystallization (Commentary) (protein specific)
Crystallization
Organism
construction of a homology model of the Papaver bracteatum SalR based on the X-ray structure of human carbonyl reductase 1, overview
Papaver bracteatum
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
F104A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
K240E
site-directed mutagenesis, inactive mutant
Papaver bracteatum
L266A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
L266S
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
L266V
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
M271T
site-directed mutagenesis, inactive mutant
Papaver bracteatum
N152A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Papaver bracteatum
N272T
site-directed mutagenesis, inactive mutant
Papaver bracteatum
R44E
site-directed mutagenesis, the mutant enzyme shows altered cofactor specificity and utilizes also NADH in contrast to the wild-type enzyme
Papaver bracteatum
R48E
site-directed mutagenesis, the mutant enzyme shows altered cofactor specificity and utilizes also NADH in contrast to the wild-type enzyme
Papaver bracteatum
S180A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Papaver bracteatum
V106A
site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme
Papaver bracteatum
Y236F
site-directed mutagenesis, inactive mutant
Papaver bracteatum
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
salutaridine
-
Papaver bracteatum
Ki Value [mM] (protein specific)
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
0.14
-
salutaridine
pH 6.0, recombinant enzyme
Papaver bracteatum
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetics of wild-type and mutant enzymes, overview
Papaver bracteatum
0.0015
-
(7S)-salutaridinol
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
0.0035
-
NADPH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
0.007
-
NADP+
pH 6.0, 40C, recombinant enzyme, with salutaridinol
Papaver bracteatum
0.0079
-
salutaridine
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
1.19
-
NADH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
34050
-
1 * 36600, recombinant His-tagged enzyme, SDS-PAGE, 1 * 34050, sequence calculation, the tertiary structure comprises the typical short-chain dehydrogenase/reductase family alpha/beta folding pattern including the four additional helices alphaF-1 to alphaF-4 assumed to prevent the dimerization, modeling and analysis, overview
Papaver bracteatum
36600
-
1 * 36600, recombinant His-tagged enzyme, SDS-PAGE, 1 * 34050, sequence calculation, the tertiary structure comprises the typical short-chain dehydrogenase/reductase family alpha/beta folding pattern including the four additional helices alphaF-1 to alphaF-4 assumed to prevent the dimerization, modeling and analysis, overview
Papaver bracteatum
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
additional information
Papaver bracteatum
the enzyme is involved in the biosynthesis of morphine
?
-
-
-
salutaridine + NADPH + H+
Papaver bracteatum
-
(7S)-salutaridinol + NADP+
-
-
r
Purification (Commentary) (protein specific)
Commentary
Organism
recombinant His-tagged enzyme from Escherichia coli by cobalt affinity chromatography
Papaver bracteatum
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
additional information
the enzyme is involved in the biosynthesis of morphine
689571
Papaver bracteatum
?
-
-
-
-
additional information
interaction of benzylisoquinoline alkaloids with enzymes, modeling, overview
689571
Papaver bracteatum
?
-
-
-
-
salutaridine + NAD(P)H + H+
NADPH is the highly preferred cofactor, the enzyme shows high substrate specificity, no activity with tropinone, (2)-menthone, codeinone and dehydroreticulinium ion, or nordehydroreticuline. Asp152, Ser180, Tyr236, and Lys240 are involved in the proton transfer system for the reduction of salutaridine, substrate-active site binding structure, overview
689571
Papaver bracteatum
(7S)-salutaridinol + NAD(P)+
the enzyme produces only the (7S)-salutaridinol stereoisomer as product, not the stereoisomer 7-epi-salutaridinol
-
-
r
salutaridine + NADPH + H+
-
689571
Papaver bracteatum
(7S)-salutaridinol + NADP+
-
-
-
r
Subunits (protein specific)
Subunits
Commentary
Organism
monomer
1 * 36600, recombinant His-tagged enzyme, SDS-PAGE, 1 * 34050, sequence calculation, the tertiary structure comprises the typical short-chain dehydrogenase/reductase family alpha/beta folding pattern including the four additional helices alphaF'-1 to alphaF'-4 assumed to prevent the dimerization, modeling and analysis, overview
Papaver bracteatum
Temperature Optimum [C] (protein specific)
Temperature Optimum [C]
Temperature Optimum Maximum [C]
Commentary
Organism
40
-
broad optimum
Papaver bracteatum
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
2.1
-
NADPH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
2.3
-
salutaridine
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
3.7
-
NADH
pH 6.0, 40C, recombinant enzyme, with salutaridine
Papaver bracteatum
21.6
-
(7S)-salutaridinol
pH 6.0, 40C, recombinant enzyme
Papaver bracteatum
21.9
-
NADP+
pH 6.0, 40C, recombinant enzyme, with salutaridinol
Papaver bracteatum
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
6
-
reduction reaction
Papaver bracteatum
9.5
-
oxidation reaction
Papaver bracteatum
pH Range (protein specific)
pH Minimum
pH Maximum
Commentary
Organism
4.5
7.5
the enzyme shows a steep decrease by 90% of activity within 1.5 pH units on both sides of the optimum at pH 6.0 for the reduction reaction
Papaver bracteatum
pI Value (protein specific)
Organism
Commentary
pI Value Maximum
pI Value
Papaver bracteatum
sequence calculation
-
4.72
Other publictions for EC 1.1.1.248
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)
720707
Wijekoon
Systematic knockdown of morphi ...
Papaver somniferum
Plant J.
69
1052-1063
2012
-
-
1
-
1
-
-
-
-
-
-
1
-
2
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
2
2
-
-
-
725460
Higashi
Atomic structure of salutaridi ...
Papaver somniferum
J. Biol. Chem.
286
6532-6541
2011
-
-
1
1
15
-
-
-
-
-
-
1
-
3
-
-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
1
2
1
15
-
-
-
-
-
-
-
-
1
-
-
-
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
3
3
-
-
-
710745
Higashi
Crystallization and preliminar ...
Papaver somniferum
Acta Crystallogr. Sect. F
66
163-166
2010
-
-
1
1
-
-
-
-
-
-
-
-
-
4
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
698937
Ziegler
Removal of substrate inhibitio ...
Papaver somniferum
J. Biol. Chem.
284
26758-26767
2009
-
-
1
-
12
-
2
26
-
-
-
-
-
1
-
-
1
-
-
-
-
-
1
-
-
-
-
13
-
-
-
1
1
-
-
-
-
1
1
-
12
-
-
2
1
26
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
13
-
-
-
-
-
-
-
-
-
-
700676
Ziegler
Evolution of morphine biosynth ...
Papaver arenarium, Papaver bracteatum, Papaver pilosum, Papaver pyrenaicum, Papaver somniferum
Phytochemistry
70
1696-1707
2009
-
-
1
-
-
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
5
-
-
-
-
-
1
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
700683
Kempe
RNAi suppression of the morphi ...
Papaver somniferum
Phytochemistry
70
579-589
2009
-
-
1
-
-
-
1
-
1
-
-
-
-
3
-
-
1
-
-
1
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
1
-
-
-
-
1
-
-
1
-
-
-
-
-
-
1
-
1
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
689571
Geissler
Molecular modeling and site-di ...
Papaver bracteatum
Plant Physiol.
143
1493-1503
2007
-
-
1
1
13
-
1
6
-
-
2
2
-
4
-
-
1
-
-
-
-
-
4
1
1
-
-
5
2
1
-
4
1
1
-
-
-
1
4
1
13
-
-
1
1
6
-
-
2
2
-
-
-
1
-
-
-
-
4
1
1
-
-
5
2
1
-
1
-
-
-
-
-
-
670562
Ziegler
Comparative transcript and alk ...
Papaver somniferum, Papaver somniferum salR
Plant J.
48
177-192
2006
-
-
1
-
-
-
-
4
-
-
3
4
-
6
-
-
1
-
-
-
-
-
8
1
1
1
-
-
2
2
-
4
-
1
-
-
-
1
4
-
-
-
-
-
-
4
-
-
3
4
-
-
-
1
-
-
-
-
8
1
1
1
-
-
2
2
-
1
-
-
-
-
-
-
9554
Gerardy
-
Purification and characterizat ...
no activity in Papaver alpinum, no activity in Papaver atlanticum, no activity in Papaver dubium, no activity in Papaver feddei, no activity in Papaver lateritium, no activity in Papaver oreophilum, no activity in Papaver orientale, no activity in Papaver persicum, no activity in Papaver pilosum, no activity in Papaver rhoeas, no activity in Papaver rupifragum, no activity in Papaver strigosum, no activity in Papaver tauricula, no activity in Papaver triniifolium, Papaver bracteatum, Papaver somniferum
Phytochemistry
34
125-132
1993
-
-
-
-
-
-
-
4
1
-
1
1
-
16
-
-
1
1
-
6
1
1
2
1
1
-
-
-
2
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4
1
-
1
1
-
-
-
1
-
6
1
1
2
1
1
-
-
-
2
2
1
-
-
-
-
-
-
-