Information on EC 1.1.1.248 - salutaridine reductase (NADPH)

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The expected taxonomic range for this enzyme is: Papaver

EC NUMBER
COMMENTARY hide
1.1.1.248
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RECOMMENDED NAME
GeneOntology No.
salutaridine reductase (NADPH)
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
salutaridinol + NADP+ = salutaridine + NADPH + H+
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
oxidation
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-
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reduction
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
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Isoquinoline alkaloid biosynthesis
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Metabolic pathways
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morphine biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
salutaridinol:NADP+ 7-oxidoreductase
Catalyses the reversible reduction of salutaridine to salutaridinol, which is a direct precursor of morphinan alkaloids in the poppy plant.
CAS REGISTRY NUMBER
COMMENTARY hide
152743-95-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
no activity in Papaver alpinum
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-
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Manually annotated by BRENDA team
no activity in Papaver atlanticum
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-
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Manually annotated by BRENDA team
no activity in Papaver dubium
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-
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Manually annotated by BRENDA team
no activity in Papaver feddei
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Manually annotated by BRENDA team
no activity in Papaver lateritium
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-
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Manually annotated by BRENDA team
no activity in Papaver oreophilum
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-
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Manually annotated by BRENDA team
no activity in Papaver orientale
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-
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Manually annotated by BRENDA team
no activity in Papaver persicum
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Manually annotated by BRENDA team
no activity in Papaver pilosum
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-
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Manually annotated by BRENDA team
no activity in Papaver rhoeas
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-
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Manually annotated by BRENDA team
no activity in Papaver rupifragum
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Manually annotated by BRENDA team
no activity in Papaver strigosum
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Manually annotated by BRENDA team
no activity in Papaver tauricula
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Manually annotated by BRENDA team
no activity in Papaver triniifolium
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Manually annotated by BRENDA team
Papaver arenarium
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
Papaver pyrenaicum
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Manually annotated by BRENDA team
gene salR; salR
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
the enzyme is a member of the short chain dehydrogenase/reductase family of enzymes. The nicotinamide moiety and the substrate-binding pocket are covered by a loop (residues 265-279), on top of which lies a large flap-like domain (residues 105-140). This configuration appears to be a combination of the two common structural themes found in other members of the short chain dehydrogenase/reductase family.
malfunction
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reduced SalR protein levels correlate with lower morphine levels and a substantial increase in the accumulation of salutaridine. Morphine biosynthesis can be perturbed at each of the six final steps
metabolism
additional information
modeling of substrate binding and conformation of bound salutaridine, interactions between SalR and its substrate and coenzyme, overview
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
salutaridine + NAD(P)H + H+
(7S)-salutaridinol + NAD(P)+
show the reaction diagram
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
the enzyme produces only the (7S)-salutaridinol stereoisomer as product, not the stereoisomer 7-epi-salutaridinol
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r
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
show the reaction diagram
salutaridine + NADPH + H+
salutaridinol + NADP+
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
show the reaction diagram
salutaridine + NADPH + H+
salutaridinol + NADP+
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
NADPH
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADP+
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product inhibition
salutaridine
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-
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0015
(7S)-salutaridinol
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pH 6.0, 40C, recombinant enzyme
1.19
NADH
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pH 6.0, 40C, recombinant enzyme, with salutaridine
0.007 - 0.198
NADP+
0.0034 - 0.125
NADPH
0.0021 - 0.4086
salutaridine
0.018
salutaridinol
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additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
21.6
(7S)-salutaridinol
Papaver bracteatum
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pH 6.0, 40C, recombinant enzyme
3.7
NADH
Papaver bracteatum
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pH 6.0, 40C, recombinant enzyme, with salutaridine
21.9
NADP+
Papaver bracteatum
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pH 6.0, 40C, recombinant enzyme, with salutaridinol
2.1
NADPH
Papaver bracteatum
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pH 6.0, 40C, recombinant enzyme, with salutaridine
0.006 - 3.188
salutaridine
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0012
NADP+
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0.14
salutaridine
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pH 6.0, recombinant enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 6
recombinant enzyme, reduction reaction
6
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reduction reaction
6 - 6.5
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formation of (7S)-salutaridinol
9 - 9.5
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formation of salutaridine
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 7.5
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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
4.7 - 7.5
sharp increase in activity at pH 4.7, half-maximal activity at pH 7.5, inactive at pH 9.0, recombinant enzyme, reduction reaction
5.8 - 7.8
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50% of maximal activity at pH 5.8 and at pH 7.8, formation of (7S)-salutaridinol
8.5 - 10
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50% of maximal activity at pH 5.8 and at pH 7.8, formationof salutaridine
9 - 10
maximal activity at pH 9.5, sharp decline in activity at pH 10.0, oxidation reaction, recombinant enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 35
40
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broad optimum
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 40
recombinant enzyme, 50% of maximal activity at 20C and 40C, reduction reaction
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.7
amino acid sequence calculation
4.72
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sequence calculation
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
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no activity in leaves
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
34050
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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
34100
1 * 34100, sequence calculation, 1 * 40000, recombinant His6-tagged enzyme, SDS-PAGE
36600
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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
40000
1 * 34100, sequence calculation, 1 * 40000, recombinant His6-tagged enzyme, SDS-PAGE
47100 - 50100
recombinant enzyme, gel filtration
52000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
construction of a homology model of the Papaver bracteatum SalR based on the X-ray structure of human carbonyl reductase 1, overview
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purified recombinant detagged wild-type and selenomethionine-substituted SalR, hanging drop vapour diffsion method, mixing of 0.002 ml of 6 mg/ml protein in 20 mM Tris buffer, pH 7.5, containing 150 mM NaCl, 5 mM 2-mercaptoethanol, and 4 mM NADPH, with 0.002 ml of reservoir solution containing 0.1 M MES, pH 6.0-6.6, 1.9 M ammonium sulfate, 5% v/v PEG 400, 0.1 M LiCl, and 3% v/v glycerol, 3 weeks, 4C, X-ray diffraction structure determination and analysis at 1.9 A resolution
to 1.9 A resolution, space group P6422 or P6222. Presence of NADPH is required for crystallization
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9
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4C, half-life: 30 days
9554
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, pH 7.0-9.0, 0.6 M KCl, half-life: 30 days
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SalR is susceptible to inactivation and soluble aggregation in an oxidative environment
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storage of purified at 193 K without 2-mercaptoethanol results in complete loss of activity. Inactive enzyme regains activity on incubation overnight at 277 K with 2-mercaptoethanol
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
His-tagged SalR purified with a cobalt affinity resin
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recombinant His-tagged enzyme from Escherichia coli by cobalt affinity chromatography
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recombinant His6-tagged enzyme from Escherichia coli strain SG13009
recombinant N-terminally His-tagged wild-type and selenomethionine-substituted SalR from Escherichia coli by cobalt affinity chromatography, cleavage of the N-terminal His-tag by thrombin, and gel filtration
to homogeneity
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into the pMyr vector and expressed as fusion protein with a myristylation sequence, which anchors the target protein to the yeast membrane. Fusion proteins (pSOS/SalAT and pMyr/SalR) coexpressed in the Saccharomyces cerevisiae cdc25H strain. SalR amplified from the recombinant plasmid SalR/pQE-30. The 0.9-kb DNA fragment ligated into an NheI/EcoRI-digested pET28a expression vector. SalR/pET28a expression construct transformed into Escherichia coli BL21(DE3)RIL
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expression in Escherichia coli
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gene salR, DNA and amino acid sequence determination and analysis, expression profile, overexpression in Escherichia coli strain SG13009 as His6-tagged enzyme
gene salR, expression of N-terminally His-tagged enzyme in Escherichia coli, a selenomethionine-substituted SalR is produced by inhibition of the methionine biosynthetic pathway with the same expression vector and Escherichia coli strain used for expression of wild-type SalR
heterologously overexpressed in its active form from Papaver somniferum
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overexpression in Escherichia coli SG13009
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real-time quantitative PCR expression analysis
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salR, DNA and amino acid sequence determination and analysis, overexpression of the His-tagged enzyme in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
F104A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
K240E
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site-directed mutagenesis, inactive mutant
L266A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
L266S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
L266V
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
M271T
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site-directed mutagenesis, inactive mutant
N152A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N272T
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site-directed mutagenesis, inactive mutant
R44E
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site-directed mutagenesis, the mutant enzyme shows altered cofactor specificity and utilizes also NADH in contrast to the wild-type enzyme
R48E
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site-directed mutagenesis, the mutant enzyme shows altered cofactor specificity and utilizes also NADH in contrast to the wild-type enzyme
S180A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
V106A
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site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme
Y236F
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site-directed mutagenesis, inactive mutant
F104A/I275A
I275V
the mutant shows altered kinetics compared to the wild-type enzyme
L185S
the mutant shows altered kinetics compared to the wild-type enzyme
L185V
the mutant shows altered kinetics compared to the wild-type enzyme
additional information
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specific virus-induced gene silencing as a functional genomics tool to investigate the regulation of morphine biosynthesis via a systematic reduction in enzyme levels responsible for the final six steps in the pathway, overview. Reduced SalR protein levels correlate with lower morphine levels and a substantial increase in the accumulation of salutaridine
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
storage of purified at 193 K without 2-mercaptoethanol results in complete loss of activity. Inactive enzyme regains activity on incubation overnight at 277 K with 2-mercaptoethanol
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