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Information on EC 1.1.1.317 - perakine reductase and Organism(s) Rauvolfia serpentina and UniProt Accession Q3L181
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1.1.1.317 perakine reductaseIUBMB Comments
The biosynthesis of raucaffrinoline from perakine is a side route of the ajmaline biosynthesis pathway. The enzyme is a member of the aldo-keto reductase enzyme superfamily from higher plants.
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The taxonomic range for the selected organisms is: Rauvolfia serpentina
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
perakine reductase, akr13d1, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
raucaffrinoline:NADP+ oxidoreductase
The biosynthesis of raucaffrinoline from perakine is a side route of the ajmaline biosynthesis pathway. The enzyme is a member of the aldo-keto reductase enzyme superfamily from higher plants.
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
4-nitroacetophenone + NADPH + H+
4-nitrobenzylalcohol + NADP+
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-
-
?
4-nitrobenzaldehyde + NADPH + H+
4-nitrobenzoate + NADP+
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-
?
perakine + NADPH + H+
raucaffrinoline + NADP+
the biosynthesis of raucaffrinoline from perakine is a side route of the ajmaline biosynthesis pathway (monoterpenoid indole alkaloid)
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-
?
perakine + NADPH + H+
raucaffrinoline + NADP+
NADPH can not be replaced by NADH. In presence of the oxidized cofactor NADP+ the reverse reaction of the reductase is low, with less than 0.5% relative activity of the forward reaction
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?
additional information
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probable dual functionality of perakine reductase, which may not only include alkaloid biosynthesis but may also include function in phenylpropanoid metabolism, generating cinnamic alcohols
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?
additional information
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the enzyme also catalyses the reduction of coniferyl aldehyde, sinapyl aldehyde, 4-coumaryl aldehyde, 3-(3,4,5-trimethoxyphenyl)propenal
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?
additional information
?
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the enzyme also catalyses the reduction of coniferyl aldehyde, sinapyl aldehyde, 4-coumaryl aldehyde, 3-(3,4,5-trimethoxyphenyl)propenal
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?
additional information
?
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enantioselective 1,2-reduction of alpha,beta-unsaturated ketones and aryl ketones by perakine reductase. Perakine reductase catalyzes asymmetric reduction of enones and aromatic ketones, leading to alpha-allylic alcohols and alpha-aromatic alcohols. It is NADPH-dependent. Among the evaluated substances, 4'-nitroacetophenone is found to be the best ketone substrate, with yield and enantiomeric excess values exceeding 99%. Exclusive enantioselectivity of enzyme perakine reductase (PR)
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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
perakine + NADPH + H+
raucaffrinoline + NADP+
the biosynthesis of raucaffrinoline from perakine is a side route of the ajmaline biosynthesis pathway (monoterpenoid indole alkaloid)
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-
?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
NADPH
dependent on, binding mode and structure, conformational changes caused by the cofactor binding, overview
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.22
4-Nitroacetophenone
recombinant enzyme, pH and temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.08
4-Nitroacetophenone
recombinant enzyme, pH and temperature not specified in the publication
29.5
4-nitrobenzaldehyde
recombinant enzyme, pH and temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
physiological function
perakine reductase catalyzes the NADPH-dependent reduction of the aldehyde perakine to yield the alcohol raucaffrinoline in the biosynthetic pathway of ajmaline in Rauvolfia, a key step in indole alkaloid biosynthesis
additional information
evolution
the enzyme belongs to the AKR13D subfamily of the aldo-keto reductases, evolutionary relationship of AKR13D from Rauvolfia serpentina to annotated AKR families and subfamilies
evolution
the enzyme is a member of aldo-ketone reductase (AKR) 13D family
additional information
the active site is formed by the catalytic tetrad Asp52, Tyr57, Lys84, and His126 at the center of the (alpha/beta)8-barrel structure. Upon NADPH binding, dramatic conformational changes and movements are observed: two additional beta-strands in the C terminus become ordered to form one alpha-helix, and a movement of up to 24 A occurs. This conformational change creates a large space that allows the binding of substrates of variable size for PR and enhances the enzyme activity
additional information
molecular modeling of the reaction mechansim. In the model, the oxygen atom of the product's hydroxy group approaches the carboxamide group of NADPH, located within a hydrogen-bond distance of the gamma-N of the imidazole ring of His126. Substrate binding analysis: the large pocket is formed by the surface of residues Ile56, Ile87, Ile 90, His126, and Arg127, the three clustered Ile residues contributing to the hydrophobic property of the pocket may facilitate substrate binding, the small pocket comprises the surface of the Met21, Tyr57, and Lys84 residues, together with the nicotinamide riboside component of NADPH
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). PERR_RAUSE
337
0
37160
Swiss-Prot
other Location (Reliability: 4)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
three-dimensional structure modeling of wild-type and mutant apo methylated enzymes with (alpha/beta)8-barrels with eight parallel beta-strands and eight alpha-helices typical for AKR superfamily members, overview
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
crystals of the purified and methylated enzyme are obtained by the hanging-drop vapour diffusion technique at 20°C with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 A, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 A
purified recombinant methylated His6-tagged enzyme wild-type and mutant A213W, hanging drop vapor diffusion method, mixing of 0.002 ml of 5.5 mg/ml protein in 10 mM Tris-HCl buffer, pH 7.0, 1 mM DTT, 10 mM EDTA, with 0002 ml reservoir solution containing 25% v/v PEG 4000, 0.1 mM sodium citrate, pH 5.6, equilibration against 1 ml of reservoir solution at 20°C for 7 days, X-ray diffraction structure determination and analysis at 2.31 A and 1.77 A resolution, respectively
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A213W
site-directed mutagenesis, cofactor-bound structure analysis
additional information
additional information
site-directed mutagenesis of each of the functional residues of catalytic tetrad Asp52, Tyr57, Lys84, His126 to an alanine residue results in more than 97.8% loss of enzyme activity
additional information
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site-directed mutagenesis of each of the functional residues of catalytic tetrad Asp52, Tyr57, Lys84, His126 to an alanine residue results in more than 97.8% loss of enzyme activity
additional information
methylation of wild-type and mutant enzymes for improvement of the crystallization process
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli by nickel affinity chromatography
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression as N-terminal His6-tagged enzyme in Escherichia coli strain M15
functionally expressed in Escherichia coli as the N-terminal His6-tagged protein
gene PR, functional recombinant expression of His-tagged enzyme in Escherichia coli
heterologous expression of a triple mutant (K98A/K242A/K294A) of perakine reductase in Escherichia coli
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Rosenthal, C.; Mueller, U.; Panjikar, S.; Sun, L.; Ruppert, M.; Zhao, Y.; Stckigt, J.
Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants
Acta Crystallogr. Sect. F
62
1286-1289
2006
Rauvolfia serpentina (Q3L181)
Sun, L.; Ruppert, M.; Sheludko, Y.; Warzecha, H.; Zhao, Y.; Stckigt, J.
Purification, cloning, functional expression and characterization of perakine reductase: the first example from the AKR enzyme family, extending the alkaloidal network of the plant Rauvolfia
Plant Mol. Biol.
67
455-467
2008
Rauvolfia serpentina (Q3L181), Rauvolfia serpentina
Sun, L.; Chen, Y.; Rajendran, C.; Mueller, U.; Panjikar, S.; Wang, M.; Mindnich, R.; Rosenthal, C.; Penning, T.M.; Stoeckigt, J.
Crystal structure of perakine reductase, founding member of a novel aldo-keto reductase (AKR) subfamily that undergoes unique conformational changes during NADPH binding
J. Biol. Chem.
287
11213-11221
2012
Rauvolfia serpentina (Q3L181)
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