Information on EC 1.1.1.317 - perakine reductase

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The expected taxonomic range for this enzyme is: Rauvolfia serpentina

EC NUMBER
COMMENTARY hide
1.1.1.317
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RECOMMENDED NAME
GeneOntology No.
perakine reductase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
raucaffrinoline + NADP+ = perakine + NADPH + H+
show the reaction diagram
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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.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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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
physiological function
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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
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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
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4-nitrobenzaldehyde + NADPH + H+
4-nitrobenzoate + NADP+
show the reaction diagram
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-
-
-
?
perakine + NADPH + H+
raucaffrinoline + 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
perakine + NADPH + H+
raucaffrinoline + NADP+
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
NADPH
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.28
4-Nitrobenzaldehyde
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pH 8.5, 37°C
0.83
perakine
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pH 8.5, 37°C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
8.26
4-Nitrobenzaldehyde
Rauvolfia serpentina
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pH 8.5, 37°C
0.014
perakine
Rauvolfia serpentina
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pH 8.5, 37°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
29.5
4-Nitrobenzaldehyde
Rauvolfia serpentina
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pH 8.5, 37°C
759
0.016
perakine
Rauvolfia serpentina
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pH 8.5, 37°C
28471
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8.5
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assay at
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8.5
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half-maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.9
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calculated from sequence
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37159
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x * 37159, calculated from sequence
38000
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x * 38000, SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
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x * 37159, calculated from sequence; x * 38000, SDS-PAGE
additional information
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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
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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
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression as N-terminal His6-tagged enzyme in Escherichia coli strain M15
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functionally expressed in Escherichia coli as the N-terminal His6-tagged protein
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heterologous expression of a triple mutant (K98A/K242A/K294A) of perakine reductase in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A213W
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site-directed mutagenesis, cofactor-bound structure analysis
D52A
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inactive mutant enzyme
H126A
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inactive mutant enzyme
K84A
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inactive mutant enzyme
Y57A
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inactive mutant enzyme
additional information
Show AA Sequence (120 entries)
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