Information on EC 2.4.1.248 - cycloisomaltooligosaccharide glucanotransferase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.4.1.248
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RECOMMENDED NAME
GeneOntology No.
cycloisomaltooligosaccharide glucanotransferase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
cyclizes part of a (1->6)-alpha-D-glucan chain by formation of a (1->6)-alpha-D-glucosidic bond
show the reaction diagram
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-
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SYSTEMATIC NAME
IUBMB Comments
(1-6)-alpha-D-glucan:(1-6)-alpha-D-glucan 6-alpha-D-[1-6alpha-D-glucano]-transferase (cyclizing)
Specific for (1->6)-alpha-D-glucans (dextrans) and, unlike cyclomaltodextrin glucanotransferase (EC 2.4.1.19), without activity towards (1->4)-alpha-D-glucans, such as amylose. It also has no activity on oligosaccharides, such as amylopectin and pullulan, containing (1->6)-alpha-D-glucosidic linkages at branch points. The enzyme from Bacillus circulans T-3040 has been shown to form cycloisomalto-oligosaccharides of three sizes (7, 8 and 9 glucose units). It will also catalyse the disproportionation of two isomalto-oligosaccharides molecules to yield a series of isomalto-oligosachharides and the addition of D-glucose to cycloisomalto-oligosaccharides with ring opening to form isomalto-oligosaccharides.
CAS REGISTRY NUMBER
COMMENTARY hide
156621-23-7
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
malfunction
physiological function
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
amylose
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
dextran
?
show the reaction diagram
glycogen
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
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yield 14.6%
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?
maltoheptaose
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
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yield 15.8%
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?
maltohexaose
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
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yield 12.7%
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?
maltopentaose
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
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yield 8.9%
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?
maltotetraose
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
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yield 4.3%
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?
maltotriose
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
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poor substrate
yield 0.9%
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?
partially hydrolyzed starch
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
S: soluble starch
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
soluble starch
cyclo-(-6)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-4)-alpha-D-Glc-(1-) + ?
show the reaction diagram
soluble starch
cyclomaltooctaose
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
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Pb2+
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1 mM, 130% of initial activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1-ethyl-3-[(3-dimethylamino)propyl]carbodiimide
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inactivation o both wild-type and mutants A452N and V744L, inactivation is reduced in presence of 10 mM Ca2+
EDTA
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1 mM, 86% residual acitvity
Fe2+
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1 mM, 94% residual acitvity
Fe3+
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1 mM, 82% residual acitvity
Hg2+
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1 mM, 55% residual acitvity
additional information
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activity of free enzyme is not influenced by NaCl up to 5 M. The activity of enzyme immobilized on Chitopearl BCW-3505 is not influenced by NaCl up to 2 M
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
additional information
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Michaelis-Menten kinetics for wild-type and deletion mutant enzymes, overview
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.1
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pH 6.0, 45
14.2
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recombinant CITase from Bacillus circulans strain G22-10, pH 5.5, 40°C
14.6
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recombinant CITase from Bacillus subtilis strain 168 DELTAaprE DELTAnprE, pH 5.5, 40°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6
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mutant MDELTA234
6.5
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mutant MDELTA23DELTA
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25
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mutant MDELTA23DELTA
30
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mutant MDELTA234
45 - 50
assay at
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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isoelectric focusing
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
102071
x * 102071, calculated
103000
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recombinant CITAse, gel filtration
106000
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2 * 106000, SDS-PAGE
184000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
?
x * 102071, calculated
dimer
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2 * 106000, SDS-PAGE
additional information
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
proteolytic modification
sequence encodes a signal peptide of 35 amino acids and a mature protein of 960 amino acids
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystal structure of the core fragment from Ser39 to Met738, devoid of its N-terminal signal peptide and C-terminal nonconserved regions. The structural model contains one catalytic (beta/alpha)8-barrel domain and three beta-domains. Domain N with an immunoglobulin-like beta-sandwich fold is attached to the N-terminus. Domain C with a Greek key beta-sandwich fold is located at the C-terminus, and a carbohydrate-binding module family 35 beta-jellyroll domain B is inserted between the 7thbeta-strand and the 7th alpha-helix of the catalytic domain A. The structures of the inactive catalytic nucleophile mutant enzyme complexed with isomaltohexaose, isomaltoheptaose, isomaltooctaose, and cycloisomaltooctaose reveal that the ligands bind in the catalytic cleft and the sugar-binding site of CBM35
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crystals of CITase bearing an N-terminal His6 tag, resolution of 2.3 A, belong to space group P3121, containing a single molecule in the asymmetric unit.. Crystals of CITase bearing a C-terminal His6 tag, resolution of 1.9 A, belong to space group P212121, containing two molecules in the asymmetric unit
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 9
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698519
5 - 10
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wild-type enzyme and mutant M123DELTA, stable at
721720
5.5 - 10
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mutant MDELTA234, stable at
721720
6 - 8.5
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mutant MDELTA23DELTA, stable at
721720
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant extracellular CITase from protease-deficient Bacillus subtilis strain 168 by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration and another step of anion exchange chromatography, recombinant extracellular CITase from Bacillus circulans G22-10 by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
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gene cit, DNA and amino acid sequence determination and analysis, expression in Escherichia coli
gene cit, expression in Escherichia coli
overexpression of CITase in Bacillus circulans strain G22-10, and, with the alpha-amylase promoter (PamyQ) and amyQ signal sequence of Bacillus amyloliquefaciens, in a protease-deficient Bacillus subtilis strain 168 as extracellular protein. The Bacillus subtilis host-vector system allows production of cycloisomaltooligosaccharides by direct fermentation of dextran along with high CITase production, which is not possible in Bacillus circulans G22-10 due to growth inhibition by dextran at high concentrations and limited production of CITase, stable expression up to 48 h
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
CITase production is induced by dextran 40, isomaltose, isomaltotriose, and panose, and soluble starch but not by G67 or dextrin
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A452N
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3fold increase in reaction velocity, 9fold increase in Km value. Activation by Ca2+ and inactivation by Cu2+ are reduced
F268V/D469Y/A513V/Y515S
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mutant produces three times as much megalo-cycloisomaltooligosaccharides (10-12 glucose units) and 1.5 times as much total cycloisomaltooligosaccharides (7-12 glucose units) as compared with the wild-type. The modified product size specificity is attributable to the construction of novel substrate-binding sites in the B-2 substrate-binding site and reactivity is improved by mutation on subsite -3 on the catalytic domain
V744L
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2fold increase in reaction velocity, 3fold increase in Km value. Activation by Ca2+ and inactivation by Cu2+ are reduced
A452N
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3fold increase in reaction velocity, 9fold increase in Km value. Activation by Ca2+ and inactivation by Cu2+ are reduced
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F268V/D469Y/A513V/Y515S
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mutant produces three times as much megalo-cycloisomaltooligosaccharides (10-12 glucose units) and 1.5 times as much total cycloisomaltooligosaccharides (7-12 glucose units) as compared with the wild-type. The modified product size specificity is attributable to the construction of novel substrate-binding sites in the B-2 substrate-binding site and reactivity is improved by mutation on subsite -3 on the catalytic domain
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V744L
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2fold increase in reaction velocity, 3fold increase in Km value. Activation by Ca2+ and inactivation by Cu2+ are reduced
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D144A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
D144N
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
D264A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D269A
site-directed mutagenesis, the mutant is inactive
D269N
site-directed mutagenesis, the mutant is almost inactive
D662A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E341A
site-directed mutagenesis, the mutant is inactive
E341Q
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
D144A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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D144N
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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D264A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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E341A
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site-directed mutagenesis, the mutant is inactive
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E341Q
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis