Information on EC 3.2.1.125 - raucaffricine beta-glucosidase

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The expected taxonomic range for this enzyme is: Rauvolfia

EC NUMBER
COMMENTARY hide
3.2.1.125
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RECOMMENDED NAME
GeneOntology No.
raucaffricine beta-glucosidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
raucaffricine + H2O = D-glucose + vomilenine
show the reaction diagram
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-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
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-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
ajmaline and sarpagine biosynthesis
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Indole alkaloid biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
raucaffricine beta-D-glucohydrolase
Highly specific; some other ajmalan glucoside alkaloids are hydrolysed, but more slowly.
CAS REGISTRY NUMBER
COMMENTARY hide
102925-37-1
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
1,2,19,20-tetrahydroraucaffricine + H2O
17-O-acetylnorajmaline + D-glucose
show the reaction diagram
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87% of activity compared to raucaffricine
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-
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1,2-(S)-dihydro-raucaffricine + H2O
2beta(R)-1,2-dihydrovomilenine + D-glucose
show the reaction diagram
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98% acivity compared to raucaffricine
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-
?
1,2-dihydro-1-methylraucaffricine + H2O
acetic acid 3-ethylidene-4-hydroxy-13-methyl-1,3,4,7,8,13,13a,13b-octahydro-2H,6H-2,7-cyclo-6,8a-methano-pyrido[1',2':1,2]azepino[3,4-b]indol-8-yl ester + D-glucose
show the reaction diagram
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36% of activity compared to raucaffricine
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-
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1,2-dihydroraucaffricine + H2O
2beta(R)-1,2-dihydrovomilenine + D-glucose
show the reaction diagram
1,2-dihydroraucaffricine + H2O
?
show the reaction diagram
recombinant RG
-
?
1-methyl-1,2-dihydroraucaffricine + H2O
?
show the reaction diagram
recombinant RG
-
?
17-O-deacetyl-1,2-dihydroraucaffricine + H2O
3-ethylidene-1,3,4,7,8,13,13a,13b-octahydro-2H,6H-2,7-cyclo-6,8a-methano-pyrido[1',2':1,2]azepino[3,4-b]indole-4,8-diol + D-glucose
show the reaction diagram
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95% of activity compared to raucaffricine
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-
?
21-glucopyranosyl-hydroxysarpagan-17-al + H2O
?
show the reaction diagram
recombinant RG
-
?
21-glucopyranosyl-hydroxysarpagan-17-ol + H2O
?
show the reaction diagram
recombinant RG
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?
5alpha-carboxystrictosidine + H2O
?
show the reaction diagram
recombinant RG
-
?
raucaffricine + H2O
D-glucose + vomilenine
show the reaction diagram
raucaffricine + H2O
vomilenine + D-glucose
show the reaction diagram
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-
-
?
secologanin + H2O
secologanin aglycone
show the reaction diagram
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0.24% acivity compared to raucaffricine
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-
?
strictosidine + H2O
?
show the reaction diagram
strictosidine + H2O
strictosidine aglycone + D-glucose
show the reaction diagram
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1.18% acivity compared to raucaffricine
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?
additional information
?
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no activity with arbutin
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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
raucaffricine + H2O
D-glucose + vomilenine
show the reaction diagram
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
D-fructose
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0.95 M: 30% inhibition
D-glucose
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0.8 M: complete inhibition
additional information
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D-glucono-1,5-lactone up to 1 M, phenylmethylsulfonyl fluoride up to 80 mM, EDTA up to 80 mM, iodoacetamide up to 80 mM, strictosidine or amygdalin up to 50 mM, mannitol up to 1 M are no inhibitors
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2
1,2,19,20-Tetrahydroraucaffricine
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1.4
1,2-Dihydro-1-methylraucaffricine
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1.5
1,2-Dihydroraucaffricine
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1.7
17-O-Deacetyl-1,2-dihydroraucaffricine
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1.3 - 1.4
Raucaffricine
1.8
strictosidine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22.5
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reaction with raucaffricine
28.1
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pH 5, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.2 - 6
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half-maximal activity at pH 4.2 and pH 6.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 55
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20°C: about 55% of maximal activity, 55°C: about 40% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
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isoelectric focusing
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60930
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sequence analysis
60931
x * 60931, sequence calculation, x * 61000, SDS-PAGE
63000 - 66000
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gel filtration
66600
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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crystallography
monomer
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in solution
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by the hanging-drop vapour diffusion technique at 20°C. Crystals reach a maximum dimension of about 0.2 * 0.15 * 0.05 mm, belong to space group I222 and diffract to 2.30 A resolution with unit-cell parameters of a = 102.8 A, b = 127.3 A and c = 215.8 A
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in complex with D-glucose
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wild type and mutant enzyme E186Q in complex with the substrate 1,2-(S)-dihydroraucaffricine and secologanin, hanging drop vapor diffusion method, using 0.01-0.3 M ammonium sulfate, 0.1 M sodium acetate, pH 4.5-5.0, 9-12% (w/v) PEG 4000 as precipitant buffer, and 20°C
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
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30 min, 50% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, crude enzyme, 30% loss of activity within 6 months
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1200fold, by Ni-NTA column
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native and recombinant RG
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
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expression in Escherichia coli
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expression in Escherichia coli TOP 10, sequencing
for heterologous expression, RG cDNA cloned into the pSE280 vector. For optimum expression, RG cDNA cloned into the pQE-2 vector and expressed in Escherichia coli strain M15
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E186D
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inactive
E186Q
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inactive
E186Q/E420Q
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inactive
E420Q
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the mutant shows 0.5% activity with raucaffricine compared to the wild type enzyme
E476A
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the mutant shows 0.57% activity with raucaffricine compared to the wild type enzyme
E476L
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the mutant shows 0.67% activity with raucaffricine compared to the wild type enzyme
F485Y
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the mutant shows 24.24% activity with raucaffricine compared to the wild type enzyme
H193A
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the mutant shows 43.66% activity with raucaffricine compared to the wild type enzyme
S390G
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the mutant shows 30.91% activity with raucaffricine compared to the wild type enzyme
T189A
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the mutant shows 63.94% activity with raucaffricine compared to the wild type enzyme
W392A
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the mutant shows 1.21% activity with raucaffricine compared to the wild type enzyme
Y200A
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the mutant shows 4.6% activity with raucaffricine compared to the wild type enzyme
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pharmacology
the RG product vomilenine is a direct intermediate in ajmaline biosynthesis and utilization of raucaffricine for the ajmaline biosynthestic pathway could be a crucial and rate-limiting step in the formation of the antiarrythmic drug ajmaline