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Information on EC 1.1.1.248 - salutaridine reductase (NADPH) and Organism(s) Papaver somniferum and UniProt Accession Q071N0
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1.1.1.248 salutaridine reductase (NADPH)IUBMB Comments
Catalyses the reversible reduction of salutaridine to salutaridinol, which is a direct precursor of morphinan alkaloids in the poppy plant.
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The taxonomic range for the selected organisms is: Papaver somniferum
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
salutaridine reductase, pssar, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
reductase, salutaridine 7-
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-
-
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salutaridine 7-reductase
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-
-
-
additional information
the enzyme belongs to the family of short-chain dehydrogenases/reductase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
salutaridinol + NADP+ = salutaridine + NADPH + H+
highly substrate specific transfer of the pro-S hydride, B-type, of NADPH to C-7 of salutaridine
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
oxidation
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-
-
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reduction
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PATHWAY SOURCE
PATHWAYS
BRENDA
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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
152743-95-8
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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
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
a step in the unique biosynthesis of morphine via codeine, overview
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-
r
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
NADPH-dependent reduction of the 7-keto group
no formation of 7-epi-salutaridinol
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r
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
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high levels of SalR expression in all Papaver somniferum varieties
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-
?
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
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interaction between SalR and SalAT in morphine biosynthesis
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-
?
salutaridine + NADPH + H+
salutaridinol + NADP+
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formation of salutaridinol, a precursor for the synthesis of morphine and related opium alkaloids
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?
additional information
?
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alkaloid composition of the organism
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?
additional information
?
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no reduction of 1,2-nordehydroreticuline, 1,2-dehydroreticulinium ion, codeinone, tropinone, or (-)-menthone, no oxidation of (R)-norreticuline, (R)-reticuline, codeine, tropine, or (+)-neomenthol
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?
additional information
?
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no reduction of 1,2-nordehydroreticuline, 1,2-dehydroreticulinium ion, codeinone, tropinone, or (-)-menthone, no oxidation of (R)-norreticuline, (R)-reticuline, codeine, tropine, or (+)-neomenthol
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-
?
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
salutaridine + NADPH + H+
(7S)-salutaridinol + NADP+
a step in the unique biosynthesis of morphine via codeine, overview
-
-
r
salutaridine + NADPH + H+
salutaridinol + NADP+
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formation of salutaridinol, a precursor for the synthesis of morphine and related opium alkaloids
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-
?
additional information
?
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alkaloid composition of the organism
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NAD+
5% of the activity with NADP+, oxidation reaction, recombinant enzyme
NADH
20% of the activity with NADPH, reduction reaction, recombinant enzyme
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
-
direct influence of a reduction in salAT transcript level by RNAi on the transcription of salR is not evident
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.023
DNA with apurinic site
pH 9.5, 30°C, recombinant enzyme, oxidation reaction
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0.031
salutaridine
pH 6.0, 30°C, recombinant enzyme, reduction reaction
additional information
additional information
kinetics, recombinant enzyme
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additional information
additional information
-
kinetics, recombinant enzyme
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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
9 - 10
maximal activity at pH 9.5, sharp decline in activity at pH 10.0, oxidation reaction, recombinant enzyme
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
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
20 - 40
recombinant enzyme, 50% of maximal activity at 20°C and 40°C, reduction reaction
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
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.
metabolism
in the biosynthetic pathway for morphine and codeine, salutaridine is reduced to salutaridinol by salutaridine reductase using NADPH as coenzyme
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
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the enzyme catalyzes a step in the morphinan alkaloid pathway, benzylisoquinoline alkaloid biosynthesis in opium poppy from (R)-reticuline to morphine overview. Morphine biosynthesis can be perturbed at each of the six final steps
additional information
additional information
modeling of substrate binding and conformation of bound salutaridine, interactions between SalR and its substrate and coenzyme, overview
additional information
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modeling of substrate binding and conformation of bound salutaridine, interactions between SalR and its substrate and coenzyme, 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). SALR_PAPSO
311
0
34050
Swiss-Prot
other Location (Reliability: 3)
PDB
SCOP
CATH
UNIPROT
ORGANISM
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
34100
1 * 34100, sequence calculation, 1 * 40000, recombinant His6-tagged enzyme, SDS-PAGE
40000
1 * 34100, sequence calculation, 1 * 40000, recombinant His6-tagged enzyme, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
monomer
1 * 34100, sequence calculation, 1 * 40000, recombinant His6-tagged enzyme, SDS-PAGE
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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, 4°C, 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
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
D107A
F104A
F104A/I275A
I275A
I275V
the mutant shows altered kinetics compared to the wild-type enzyme
K186V
L185A
L185S
the mutant shows altered kinetics compared to the wild-type enzyme
L185V
the mutant shows altered kinetics compared to the wild-type enzyme
L266A
M271A
N272A
S181A
T182A
V106A
additional information
-
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
D107A
the mutant shows altered kinetics compared to the wild-type enzyme
F104A
the mutant shows altered kinetics compared to the wild-type enzyme
F104A/I275A
shows no substrate inhibition accompanied by a weak affinity for productive salutaridine binding, kcat is almost 2fold higher compared with the wild-type
F104A/I275A
substitution of Phe104 in the substrate-binding pocket, and Ile275 under the flap domain, the mutant shows altered kinetics compared to the wild-type enzyme
I275A
increase in Km, shows an increase of velocity by a factor of 2.3
I275A
the mutant shows altered kinetics compared to the wild-type enzyme
K186V
moderate effects on substrate affinity, exhibits a turnover number similar to that of the wild-type
K186V
the mutant shows altered kinetics compared to the wild-type enzyme
L185A
the mutant shows altered kinetics compared to the wild-type enzyme
L266A
the mutant shows altered kinetics compared to the wild-type enzyme
M271A
the mutant shows altered kinetics compared to the wild-type enzyme
N272A
the mutant shows altered kinetics compared to the wild-type enzyme
S181A
moderate effects on substrate affinity, increase in kcat by 50%
S181A
the mutant shows altered kinetics compared to the wild-type enzyme
T182A
the mutant shows altered kinetics compared to the wild-type enzyme
V106A
moderate effects on substrate affinity, increase in kcat by 90%
V106A
the mutant shows altered kinetics compared to the wild-type enzyme
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
SalR is susceptible to inactivation and soluble aggregation in an oxidative environment
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
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
salutaridine synthase (PsSAS), salutaridine reductase (PsSAR) and salutaridinol acetyltransferase (PsSAT) are functionally coexpressed in Saccharomyces cerevisiae and optimization of the pH conditions allowed for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. A 7-gene pathway for the production of codeine and morphine from (R)-reticuline is reconstituted. Yeast cell feeding assays using (R)-reticuline, salutaridine or codeine as substrates show that all enzymes are functionally coexpressed in yeast and that activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis. The results of this study describe a significant advance for the synthesis of morphinans in Saccharomyces cerevisiae and pave the way for their complete synthesis in recombinant microbes
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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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
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
KP400665
the Papaper somniferum enzymes salutaridine synthase (SAS), salutaridine reductase (SAR) and salutaridinol acetyltransferase (SAT) are functionally co-expressed in Saccharomyces cerevisiae and optimization of the pH conditions allows for productive spontaneous rearrangement of salutaridinol-7-O-acetate and synthesis of thebaine from (R)-reticuline. Upon reconstitution of a 7-gene pathway for the production of codeine and morphine from (R)-reticuline, activity of salutaridine reductase and codeine-O-demethylase likely limit flux to morphine synthesis
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Gerardy, R.; Zenk, M.H.
Purification and characterization of salutaridine:NADPH 7-oxidoreductase from Papaver somniferum
Phytochemistry
34
125-132
1993
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
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Ziegler, J.; Voigtlaender, S.; Schmidt, J.; Kramell, R.; Miersch, O.; Ammer, C.; Gesell, A.; Kutchan, T.M.
Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis
Plant J.
48
177-192
2006
Papaver somniferum (Q071N0), Papaver somniferum, Papaver somniferum salR (Q071N0)
Ziegler, J.; Brandt, W.; Geissler, R.; Facchini, P.J.
Removal of substrate inhibition and increase in maximal velocity in the short chain dehydrogenase/reductase salutaridine reductase involved in morphine biosynthesis
J. Biol. Chem.
284
26758-26767
2009
Papaver somniferum (Q071N0)
Ziegler, J.; Facchini, P.J.; Geissler, R.; Schmidt, J.; Ammer, C.; Kramell, R.; Voigtlaender, S.; Gesell, A.; Pienkny, S.; Brandt, W.
Evolution of morphine biosynthesis in opium poppy
Phytochemistry
70
1696-1707
2009
Papaver arenarium, Papaver bracteatum (A4UHT7), Papaver pilosum, Papaver pyrenaicum, Papaver somniferum
Kempe, K.; Higashi, Y.; Frick, S.; Sabarna, K.; Kutchan, T.M.
RNAi suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes
Phytochemistry
70
579-589
2009
Papaver somniferum
Higashi, Y.; Smith, T.J.; Jez, J.M.; Kutchan, T.M.
Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from the opium poppy Papaver somniferum
Acta Crystallogr. Sect. F
66
163-166
2010
Papaver somniferum (Q071N0), Papaver somniferum
Wijekoon, C.P.; Facchini, P.J.
Systematic knockdown of morphine pathway enzymes in opium poppy using virus-induced gene silencing
Plant J.
69
1052-1063
2012
Papaver somniferum
Higashi, Y.; Kutchan, T.M.; Smith, T.J.
Atomic structure of salutaridine reductase from the opium poppy (Papaver somniferum)
J. Biol. Chem.
286
6532-6541
2011
Papaver somniferum (Q071N0), Papaver somniferum
Fossati, E.; Narcross, L.; Ekins, A.; Falgueyret, J.P.; Martin, V.J.
Synthesis of morphinan alkaloids in Saccharomyces cerevisiae
PLoS ONE
10
e0124459
2015
Papaver somniferum (KP400665), Papaver somniferum
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