Information on EC 2.4.1.9 - inulosucrase

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The expected taxonomic range for this enzyme is: Eukaryota, Bacteria

EC NUMBER
COMMENTARY hide
2.4.1.9
-
RECOMMENDED NAME
GeneOntology No.
inulosucrase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
sucrose + [(2->1)-beta-D-fructosyl]n = glucose + [(2->1)-beta-D-fructosyl]n+1
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
fructosyl group transfer
-
-
-
-
hexosyl group transfer
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Starch and sucrose metabolism
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-
SYSTEMATIC NAME
IUBMB Comments
sucrose:(2->1)-beta-D-fructan 1-beta-D-fructosyltransferase
Converts sucrose into inulin and D-glucose. Some other sugars can act as D-fructosyl acceptors.
CAS REGISTRY NUMBER
COMMENTARY hide
9030-16-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain JN19
-
-
Manually annotated by BRENDA team
strain CRF 202
-
-
Manually annotated by BRENDA team
strain CRF 202
-
-
Manually annotated by BRENDA team
strain CFR77
-
-
Manually annotated by BRENDA team
strain KCCM12017
-
-
Manually annotated by BRENDA team
strain ATCC 20524
-
-
Manually annotated by BRENDA team
strain NCC 533
UniProt
Manually annotated by BRENDA team
strain TMW1.106
-
-
Manually annotated by BRENDA team
strain CW2. Enzyme is a multidomain fructosyltrasferase containing domains from glucosyltransferase
UniProt
Manually annotated by BRENDA team
strain CW28, isolated from Pozol, a traditional lactic acid fermented beverage of Mayan origin produced from lime treated corn in the south of mexico
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-
Manually annotated by BRENDA team
strain ATCC 28811
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-
Manually annotated by BRENDA team
strain FERM P-15944
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-
Manually annotated by BRENDA team
strain FERM P-15944
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-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
3 sucrose + sucrose
3 alpha-D-glucose + (2,1-beta-D-fructosyl)n+1
show the reaction diagram
raffinose + [(2->1)-beta-D-fructosyl]n
glucose + [(2->1)-beta-D-fructosyl]n+1
show the reaction diagram
sucrose + (2,1-beta-D-fructosyl)n
alpha-D-glucose + (2,1-beta-D-fructosyl)n+1
show the reaction diagram
sucrose + (2,1-beta-D-fructosyl)n
D-glucose + (2,1-beta-D-fructosyl)n+1
show the reaction diagram
sucrose + H2O
alpha-D-glucose + beta-D-fructose
show the reaction diagram
sucrose + H2O
D-fructose + D-glucose
show the reaction diagram
sucrose + hydroquinone
?
show the reaction diagram
-
-
-
-
?
sucrose + kestose
?
show the reaction diagram
sucrose + melibiose
glucose + ?
show the reaction diagram
sucrose + raffinose
?
show the reaction diagram
sucrose + raffinose
glucose + 1-kestose
show the reaction diagram
sucrose + sucrose
kestose + nystose + fructooligosaccharides
show the reaction diagram
sucrose + sucrose
[(beta-D-fructose-(2-1))n]-alpha-D-glucose + alpha-D-glucose
show the reaction diagram
sucrose + sucrose
[(beta-D-Fruf-(2-1))5]-alpha-D-Glup + alpha-D-glucose
show the reaction diagram
sucrose + [(2->1)-beta-D-fructosyl]n
glucose + [(2->1)-beta-D-fructosyl]n+1
show the reaction diagram
sucrose + [(beta-D-Fruf-(2-1))n]-alpha-D-Glup
alpha-D-glucose + [(beta-D-Fruf-(2-1))n+1]-alpha-D-Glup
show the reaction diagram
additional information
?
-
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
3 sucrose + sucrose
3 alpha-D-glucose + (2,1-beta-D-fructosyl)n+1
show the reaction diagram
-
-
-
-
?
sucrose + (2,1-beta-D-fructosyl)n
alpha-D-glucose + (2,1-beta-D-fructosyl)n+1
show the reaction diagram
sucrose + (2,1-beta-D-fructosyl)n
D-glucose + (2,1-beta-D-fructosyl)n+1
show the reaction diagram
sucrose + [(2->1)-beta-D-fructosyl]n
glucose + [(2->1)-beta-D-fructosyl]n+1
show the reaction diagram
sucrose + [(beta-D-Fruf-(2-1))n]-alpha-D-Glup
alpha-D-glucose + [(beta-D-Fruf-(2-1))n+1]-alpha-D-Glup
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Fe2+
-
slightly enhances activity
K+
-
slightly enhances activity
Mg2+
-
slightly enhances activity
additional information
-
no effect by Mg2+, K+, and Na+ at 1 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cu2+
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98% inhibition at 1 mM
Fe2+
-
54% inhibition at 1 mM
Fe3+
-
72% inhibition at 1 mM
Hg2+
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97% inhibition at 1 mM
Zn2+
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91% inhibition at 1 mM
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(NH4)NO3
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10 mM, 15fold enhancement of activity
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
11.4 - 308
Sucrose
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.3 - 504
Sucrose
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.019 - 0.861
Sucrose
55
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.00032
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purified recombinant mutant D272N, inulin synthesizing activity
0.00066
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purified recombinant mutant D424N, inulin synthesizing activity
0.0028
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purified recombinant mutant E523Q, inulin synthesizing activity
0.3
below, purified recombinant mutant D520A, 50C, without Ca2+
1
purified recombinant mutant D520N, 50C, 1 mM Ca2+
3.6
purified recombinant mutant D520A, 50C, 1 mM Ca2+
10.5
purified recombinant truncated His-tagged enzyme, pH 5.2, 55C
27
hydrolytic activity, 55C, pH 7.0
47
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purified recombinant wild-type enzyme, inulin synthesizing activity
178
hydrolytic activity, 37C, pH 7.0
180
purified recombinant wild-type enzyme, 50C, 1 mM Ca2+
187
transglycosylation activity, 37C, pH 7.0
365
total activity, 37C, pH 7.0
459
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pH 5.5, 55C
698
transglycosylation activity, 55C, pH 7.0
719
total activity, 55C, pH 7.0
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 6.5
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crude extract
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.7 - 6.5
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50% activity at pH 3.7 an pH 6.5
4.5 - 6
more than 85% of maximum activity
5 - 10
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almost no activity below pH 5 and above pH 10.0
7.5
sharp decrease in activity above
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
35
both wild-type and truncated version IslA2
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 60
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transferase activity: 20% activity at 20C, 90% activity at 60C, rapid decrease in activity above 60C, inactive at 70C
22 - 55
in presence of Ca2+ at 1 mM: optimal activity at 55C, in absence of Ca2+ and EDTA: decrease in activity above 45C, completely inactive at 55C, overview
60
sharp decrease in activity above
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
PDB
SCOP
CATH
ORGANISM
UNIPROT
Lactobacillus johnsonii (strain CNCM I-12250 / La1 / NCC 533)
Lactobacillus johnsonii (strain CNCM I-12250 / La1 / NCC 533)
Lactobacillus johnsonii (strain CNCM I-12250 / La1 / NCC 533)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
116300
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estimated from SDS-PAGE
165000
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x * 165000, SDS-PAGE
570000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant C-terminally His-tagged inulosucrase residues 145-708 fragment, X-ray diffraction structure determination and analysis at 1.75 A resolution, molecular replacement
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 7
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702814
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
Ca2+ stabilizes the enzyme
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20C, purified recombinant undiluted enzyme, 10% glycerol, completely stable for at least several months
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4C, purified recombinant enzyme, undiluted stable for several days, 10times diluted stable for more than a month
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
partial
recombinant enzyme
recombinant His-tagged full-length wild-type enzyme and truncated mutant enzyme from Escherichia coli strain BL21 by nickel affinity chromatography and ultrafiltration
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli TOP10 cells by nickel affinity chromatography
recombinant His6-tagged truncated enzyme mutant from Escherichia coli strain DH10B by nickel affinity chromatography
recombinant Myc/His-tagged enzyme from Escherichia coli
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recombinant Myc/His-tagged wild-type and mutant enzymes from Escherichia coli
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recombinant Myc/His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
expression of His-tagged wild-type and mutant enzymes in Escherichia coli TOP10 cells
expression of Myc/His-tagged enzyme in Escherichia coli
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expression of Myc/His-tagged wild-type and mutant enzymes in Escherichia coli
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expression of the full-length enzyme and two C-terminally truncated enzyme forms in Escherichia coli. Natural chimeric enzyme with three domains: the first and third with high identity with alternansucrase, the second domain, which is the catalytic one has similarity with levansucrase and inulosucrase
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expression of the full-length protein and a truncated protein with a 100 amino acid truncation at the C-terminus from amino acid 699 onwards, with a C-terminal His-tag, InuDELTA699His
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gene inuGB, recombinant expression of His6-tagged truncated enzyme mutant lacking the cell-anchoring motif in Escherichia coli strain DH10B
recombinant expression of C-terminally His-tagged fragment of inulosucrase comprising residues 145-708
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recombinant expression of His-tagged full-length wild-type enzyme and truncated mutant enzyme in Escherichia coli strain BL21
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recombinant overexpression of the enzyme lacking the cell-anchoring motif in Escherichia coli
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the truncated mutant IslA4 is expressed in Escherichia coli BL21(DE3) cells
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
N301A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N301S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N305A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N305S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
N301A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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N301S
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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N305A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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N305S
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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A417N
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme
A425P
site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
A425P/A538S/N543S/D548R/W551T
site-directed mutagenesis, the mutant shows 65% reduced activity compared to the wild-type enzyme
A489G
site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
A538S
site-directed mutagenesis, the mutation, located behind the general acid/base, increases the enzyme activity two to threefold
D272N
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
D424N
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
D520A
site-directed mutagenesis, reduced activity in absence of Ca2+ at 30C, reduction in affinity for Ca2+ at higher temperatures, overview
D520N
site-directed mutagenesis, mutant enzyme is nearly inactive in presence of 1 mM Ca2+ and completely inactive in absence of Ca2+
D548R
site-directed mutagenesis, increased activity compared to the wild-type enzyme
E523Q
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
G416E
site-directed mutagenesis, the mutation at the rim of the active site pocket in loop 415-423, increases the hydrolytic activity twofold, without significantly changing the transglycosylation activity
K415R
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme
N365K
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
N543S
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme; site-directed mutagenesis, the mutation, located adjacent to the +1/+2 subsite residue R544, results in synthesis of a reduced variety of fructooligosaccharides compared to the wild-type enzyme
P516L
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme
R423H
-
inactive mutant enzyme
R423K
-
altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer than wild-type enzyme. KM-value for sucrose is 3.3fold higher than wild-type value. Vmax is 28.8fold lower than wild-type value
S442N
site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
T366L
site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
T366L/A425P/N365K
site-directed mutagenesis, the mutant shows 47% reduced activity compared to the wild-type enzyme
T413K
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
T413K/K415R/G416E/A425P/S442N/W486L/P516L
site-directed mutagenesis, the mutant synthesizes 1-kestose only, but at low efficiency, it shows 94% reduced activity compared to the wild-type enzyme
W271N
-
altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer than wild-type enzyme. KM-value for sucrose is 15.6fold higher than wild-type value. Vmax is 16.9fold lower than wild-type value
W340N
-
inactive mutant enzyme
W486L
site-directed mutagenesis, the mutant shows increased activity compared to the wild-type enzyme
W551T
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
A425P
-
site-directed mutagenesis, slightly reduced activity compared to the wild-type enzyme
-
D272N
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
-
D424N
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
-
D520A
-
site-directed mutagenesis, reduced activity in absence of Ca2+ at 30C, reduction in affinity for Ca2+ at higher temperatures, overview
-
D520N
-
site-directed mutagenesis, mutant enzyme is nearly inactive in presence of 1 mM Ca2+ and completely inactive in absence of Ca2+
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E523Q
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
-
G416E
-
site-directed mutagenesis, the mutation at the rim of the active site pocket in loop 415-423, increases the hydrolytic activity twofold, without significantly changing the transglycosylation activity
-
K415R
-
site-directed mutagenesis, unaltered activity compared to the wild-type enzyme
-
R423H
-
inactive mutant enzyme
-
R423K
-
altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer than wild-type enzyme. KM-value for sucrose is 3.3fold higher than wild-type value. Vmax is 28.8fold lower than wild-type value
-
T413K
-
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
-
W271N
-
altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer than wild-type enzyme. KM-value for sucrose is 15.6fold higher than wild-type value. Vmax is 16.9fold lower than wild-type value
-
W340N
-
inactive mutant enzyme
-
L499F
-
the mutation reduces the affinity for sucrose 3fold
R618K
-
mutant shows decreased activity compared to the wild type enzyme
R696K
-
the mutant only produces inulin
S425A
-
the mutant displays complex kinetic behavior in which the transfructosylation rate is described by first-order kinetics, while sucrose hydrolysis follows Michaelis-Menten behavior, the hydrolytic activity of S425A is reduced to about 52% of the converted sucrose; the mutant no longer produces high-molecular-weight inulin
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology