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Information on EC 3.2.1.125 - raucaffricine beta-glucosidase and Organism(s) Rauvolfia serpentina and UniProt Accession Q9SPP9

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IUBMB Comments
Highly specific; some other ajmalan glucoside alkaloids are hydrolysed, but more slowly.
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This record set is specific for:
Rauvolfia serpentina
UNIPROT: Q9SPP9
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Word Map
The taxonomic range for the selected organisms is: Rauvolfia serpentina
The enzyme appears in selected viruses and cellular organisms
Synonyms
raucaffricine-o-beta-d-glucosidase, raucaffricine glucosidase, raucaffricine beta-glucosidase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
raucaffricine glucosidase
-
raucaffricine-O-beta-D-glucosidase
-
glucosidase, raucaffricine beta-
-
-
-
-
raucaffricine beta-D-glucosidase
-
-
-
-
raucaffricine glucosidase
raucaffricine O-beta-D-glucosidase
-
-
additional information
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
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
-
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-dihydroraucaffricine + H2O
?
show the reaction diagram
recombinant RG
-
?
1-methyl-1,2-dihydroraucaffricine + H2O
?
show the reaction diagram
recombinant RG
-
?
21-glucopyranosyl-hydroxysarpagan-17-al + H2O
?
show the reaction diagram
recombinant RG
-
?
21-glucopyranosyl-hydroxysarpagan-17-ol + H2O
?
show the reaction diagram
recombinant RG
-
?
5alpha-carboxystrictosidine + H2O
?
show the reaction diagram
recombinant RG
-
?
raucaffricine + H2O
D-glucose + vomilenine
show the reaction diagram
strictosidine + H2O
?
show the reaction diagram
only recombinant RG, no substrate of native RG
-
?
1,2,19,20-tetrahydroraucaffricine + H2O
17-O-acetylnorajmaline + D-glucose
show the reaction diagram
-
87% of activity compared to raucaffricine
-
-
?
1,2-(S)-dihydro-raucaffricine + H2O
2beta(R)-1,2-dihydrovomilenine + D-glucose
show the reaction diagram
-
98% acivity compared to raucaffricine
-
-
?
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
-
36% of activity compared to raucaffricine
-
-
?
1,2-dihydroraucaffricine + H2O
2beta(R)-1,2-dihydrovomilenine + D-glucose
show the reaction diagram
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
-
95% of activity compared to raucaffricine
-
-
?
raucaffricine + H2O
D-glucose + vomilenine
show the reaction diagram
raucaffricine + H2O
vomilenine + D-glucose
show the reaction diagram
-
-
-
?
secologanin + H2O
secologanin aglycone
show the reaction diagram
-
0.24% acivity compared to raucaffricine
-
-
?
strictosidine + H2O
?
show the reaction diagram
-
-
-
-
?
strictosidine + H2O
strictosidine aglycone + D-glucose
show the reaction diagram
-
1.18% acivity compared to raucaffricine
-
-
?
additional information
?
-
-
no activity with arbutin
-
-
?
NATURAL SUBSTRATE
NATURAL PRODUCT
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
raucaffricine + H2O
D-glucose + vomilenine
show the reaction diagram
-
involved in the biosynthesis of alkaloids, e.g. ajmaline
-
?
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
N-(bromobenzyl)-beta-D-gluco-1,5-deoxa-pyranosylamine
the inhibitor anchors exclusively in the catalytic active site by competition with appropriate enzyme substrates
N-(cyclohexyl)-beta-D-gluco-1,5-deoxa-pyranosylamine
the inhibitor anchors exclusively in the catalytic active site by competition with appropriate enzyme substrates
N-(cyclohexylmethyl)-beta-D-gluco-1,5-deoxa-pyranosylamine
the inhibitor anchors exclusively in the catalytic active site by competition with appropriate enzyme substrates
D-fructose
-
0.95 M: 30% inhibition
D-glucose
-
0.8 M: complete inhibition
additional information
-
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
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3
Raucaffricine
pH 5, 28°C, recombinant RG
1.8
strictosidine
pH 5, 28°C, recombinant RG
2
1,2,19,20-Tetrahydroraucaffricine
-
-
1.4
1,2-Dihydro-1-methylraucaffricine
-
-
1.5
1,2-Dihydroraucaffricine
-
-
1.7
17-O-Deacetyl-1,2-dihydroraucaffricine
-
-
1.3 - 1.4
Raucaffricine
1.8
strictosidine
-
wild type enzyme, pH and temperature not specified in the publication
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.000066
N-(bromobenzyl)-beta-D-gluco-1,5-deoxa-pyranosylamine
30°C, pH 5.0
0.628
N-(cyclohexyl)-beta-D-gluco-1,5-deoxa-pyranosylamine
30°C, pH 5.0
0.00022
N-(cyclohexylmethyl)-beta-D-gluco-1,5-deoxa-pyranosylamine
30°C, pH 5.0
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
22.5
-
reaction with raucaffricine
28.1
-
pH 5, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.2 - 6
-
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
-
20°C: about 55% of maximal activity, 55°C: about 40% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.8
-
isoelectric focusing
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
RG1_RAUSE
540
0
60934
Swiss-Prot
other Location (Reliability: 3)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60931
x * 60931, sequence calculation, x * 61000, SDS-PAGE
61000
x * 60931, sequence calculation, x * 61000, SDS-PAGE
60930
-
sequence analysis
61000
63000 - 66000
-
gel filtration
66600
-
gel filtration
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 60931, sequence calculation, x * 61000, SDS-PAGE
?
-
x * 61000, SDS-PAGE
dimer
-
crystallography
monomer
-
in solution
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
no glycoprotein
-
-
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with the (1R,2S,3S,4R,5R)-4-(cyclohexylmethylamino)-5-(hydroxymethyl)cyclopentane-1,2,3-triol
in complex with D-glucose
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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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
-
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E186D
-
inactive
E186Q
-
inactive
E186Q/E420Q
-
inactive
E420Q
-
the mutant shows 0.5% activity with raucaffricine compared to the wild type enzyme
E476A
-
the mutant shows 0.57% activity with raucaffricine compared to the wild type enzyme
E476L
-
the mutant shows 0.67% activity with raucaffricine compared to the wild type enzyme
F485Y
-
the mutant shows 24.24% activity with raucaffricine compared to the wild type enzyme
H193A
-
the mutant shows 43.66% activity with raucaffricine compared to the wild type enzyme
S390G
-
the mutant shows 30.91% activity with raucaffricine compared to the wild type enzyme
T189A
-
the mutant shows 63.94% activity with raucaffricine compared to the wild type enzyme
W392A
-
the mutant shows 1.21% activity with raucaffricine compared to the wild type enzyme
Y200A
-
the mutant shows 4.6% activity with raucaffricine compared to the wild type enzyme
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
55
-
30 min, 50% loss of activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4°C, crude enzyme, 30% loss of activity within 6 months
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native and recombinant RG
1200fold, by Ni-NTA column
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli
expression in Escherichia coli
expression in Escherichia coli TOP 10, sequencing
heterologously expressed in Escherichia coli
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
-
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
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Schuebel, H.; Stoeckigt, J.; Feicht, R.; Simon, H.
Partial purification and characterization of raucaffricine beta-D-glucosidase from plant cell-suspension cultures of Rauwolfia serpentina Benth
Helv. Chim. Acta
69
538-547
1986
Rauvolfia caffra, Rauvolfia mannii, Rauvolfia serpentina, Rauvolfia verticillata
-
Manually annotated by BRENDA team
Warzecha, H.; Obitz, P.; Stockigt, J.
Purification, partial amino acid sequence and structure of the product of raucaffricine-O-beta-D-glucosidase from plant cell cultures of Rauwolfia serpentina
Phytochemistry
50
1099-1109
1999
Rauvolfia serpentina
Manually annotated by BRENDA team
Warzecha, H.; Gerasimenko, I.; Kutchan, T.M.; Stockigt, J.
Molecular cloning and functional bacterial expression of a plant glucosidase specifically involved in alkaloid biosynthesis
Phytochemistry
54
657-666
2000
Rauvolfia serpentina (Q9SPP9), Rauvolfia serpentina
Manually annotated by BRENDA team
Ruppert, M.; Panjikar, S.; Barleben, L.; Stckigt, J.
Heterologous expression, purification, crystallization and preliminary x-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis
Acta Crystallogr. Sect. F
62
257-260
2006
Rauvolfia serpentina (Q9SPP9), Rauvolfia serpentina
Manually annotated by BRENDA team
Stoeckigt, J.; Panjikar, S.; Ruppert, M.; Barleben, L.; Ma, X.; Loris, E.; Hill, M.
The molecular architecture of major enzymes from ajmaline biosynthetic pathway
Phytochem. Rev.
6
15-34
2007
Rauvolfia serpentina
-
Manually annotated by BRENDA team
Xia, L.; Ruppert, M.; Wang, M.; Panjikar, S.; Lin, H.; Rajendran, C.; Barleben, L.; Stoeckigt, J.
Structures of alkaloid biosynthetic glucosidases decode substrate specificity
ACS Chem. Biol.
7
226-234
2012
Rauvolfia serpentina
Manually annotated by BRENDA team
Xia, L.; Rajendran, C.; Ruppert, M.; Panjikar, S.; Wang, M.; Stoeckigt, J.
High speed X-ray analysis of plant enzymes at room temperature
Phytochemistry
91
88-92
2013
Rauvolfia serpentina (Q9SPP9)
Manually annotated by BRENDA team
Xia, L.; Lin, H.; Staniek, A.; Panjikar, S.; Ruppert, M.; Hilgers, P.; Williardt, J.; Rajendran, C.; Wang, M.; Warzecha, H.; Jger, V.; Stckigt, J.
Ligand structures of synthetic deoxa-pyranosylamines with raucaffricine and strictosidine glucosidases provide structural insights into their binding and inhibitory behaviours
J. Enzyme Inhib. Med. Chem.
30
472-478
2015
Rauvolfia serpentina (Q9SPP9)
Manually annotated by BRENDA team