Information on EC 3.2.1.57 - isopullulanase

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

EC NUMBER
COMMENTARY
3.2.1.57
-
RECOMMENDED NAME
GeneOntology No.
isopullulanase
-
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of O-glycosyl bond
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
pullulan 4-glucanohydrolase (isopanose-forming)
The enzyme has practically no action on starch. Panose (4-alpha-isomaltosylglucose) is hydrolysed to isomaltose and glucose. cf. EC 3.2.1.41 (pullulanase) and EC 3.2.1.135 (neopullulanase).
SYNONYMS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IPU
Aspergillus brasiliensis ATCC 9642
-
-
pullulan 4-glucanohydrolase
-
-
IPU
-
-
-
additional information
-
classified under pullulanase type II
additional information
-
classified under pullulanase type II
-
CAS REGISTRY NUMBER
COMMENTARY
37288-43-0
-
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
T6, single enzyme with G2-dextranase EC 3.2.1.94 and isopullanase activity
-
-
Manually annotated by BRENDA team
formerly Aspergillus niger ATCC 9642
UniProt
Manually annotated by BRENDA team
Aspergillus brasiliensis ATCC 9642
formerly Aspergillus niger ATCC 9642
UniProt
Manually annotated by BRENDA team
ATCC 9642, IPU F1 and IPU F2
-
-
Manually annotated by BRENDA team
ATCC9642
Uniprot
Manually annotated by BRENDA team
strain ATCC 9642
-
-
Manually annotated by BRENDA team
Aspergillus niger ATCC9642
ATCC9642
Uniprot
Manually annotated by BRENDA team
strain US149, likely belonging to Bacillus naganoensis
-
-
Manually annotated by BRENDA team
strain US149, likely belonging to Bacillus naganoensis
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
the enzyme belongs to the glycoside hydrolase family 49 (GH49)
evolution
Aspergillus brasiliensis ATCC 9642
-
the enzyme belongs to the glycoside hydrolase family 49 (GH49)
-
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
4(2)-alpha-isomaltosylisomaltose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
4(3)-alpha-panosylpanose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(2)-alpha-isomaltosylmaltose + H2O
?
show the reaction diagram
-
isomaltotriosylglucose, cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(2)-alpha-maltosylmaltose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
6(3)-alpha-glucosylmaltotriose + H2O
?
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage of the panose motif
-
-
?
alpha-glucosylmaltotriose + H2O
isomaltose + maltose
show the reaction diagram
-
-
-
?
alpha-maltosylmaltose + H2O
isopanose + glucose
show the reaction diagram
-
-
-
?
alpha-maltosylmaltose + H2O
isopanose + glucose
show the reaction diagram
-
-
-
?
panose + H2O
?
show the reaction diagram
-
-
-
-
?
panose + H2O
isomaltose + glucose
show the reaction diagram
-
-
-
?
panose + H2O
isomaltose + glucose
show the reaction diagram
-
-
-
?
panose + H2O
isomaltose + glucose
show the reaction diagram
-
-
-
?
panose + H2O
isomaltose + glucose
show the reaction diagram
-
-
-
?
panose + H2O
isomaltose + glucose
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
small amount
?
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
Arthrobacter globiformis T6
-
-
-
?
pullulan + H2O
isopanose
show the reaction diagram
-
cleaves the alpha-1,4-glycosidic linkage
-
?
pullulan + H2O
isopanose
show the reaction diagram
-
IPU degrades alpha-1,4-glycosidic linkage from the reducing end adjacent to alpha-1,6-glycosidic linkage in pullulan
-
?
pullulan + H2O
isopanose
show the reaction diagram
-
very strict substrate specificity, preferred over panose
-
?
pullulan + H2O
isopanose
show the reaction diagram
the enzyme cleaves the alpha(1,4) glucosidic linkage using an inverting mechanism and liberating D-glucose
trisaccharide [Glc-alpha(1,4)-Glc-alpha(1,6)-Glc]
?
pullulan + H2O
isopanose
show the reaction diagram
-
IPU degrades alpha-1,4-glycosidic linkage from the reducing end adjacent to alpha-1,6-glycosidic linkage in pullulan
-
?
pullulan + H2O
isopanose
show the reaction diagram
Aspergillus brasiliensis ATCC 9642
the enzyme cleaves the alpha(1,4) glucosidic linkage using an inverting mechanism and liberating D-glucose
trisaccharide [Glc-alpha(1,4)-Glc-alpha(1,6)-Glc]
?
pullulan + H2O
isopanose + ?
show the reaction diagram
-
-
-
-
pullulan + H2O
isopanose + ?
show the reaction diagram
Asp353, Asp372 and Asp373 are the catalytic residues of IPU
-
?
azidopullulan + H2O
?
show the reaction diagram
-
-
-
-
?
additional information
?
-
-
not: amylose, maltoheptaose, starch, amylopectin
-
-
-
additional information
?
-
-
recombinant IPU hydrolyzes various substrates containing the structure of panose indicating a strict subsite recognition of the panose motif, cleaves the alpha-1,4-glycosidic linkage
-
-
-
additional information
?
-
-
not: amylose, maltoheptaose, starch, amylopectin
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)4
show the reaction diagram
-
-
small amount
?
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
-
-
-
?
pullulan + H2O
isopanose + (Glc)6
show the reaction diagram
Arthrobacter globiformis T6
-
-
-
?
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ag+
-
5 mM, partial inhibition
Fe3+
-
5 mM completeinactivation
Hg2+
-
5 mM complete inactivation
KMnO4
-
5 mM complete inactivation
N-bromosuccinimide
-
5 mM complete inactivation
N-bromosuccinimide
-
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
39.2
panose
-
40C, recombinant IPU
160
panose
-
pH 3.5, 40C, wild-type enzyme
920
panose
-
pH 3.5, 40C, mutant enzyme W402F
0.88
Pullulan
-
40C, recombinant IPU
additional information
additional information
-
kinetic parameters for deglycosylated recombinant IPU with a sugar content of 13.8%, 6.8% and 2.1%, respectively, deglycosylation decreases the kinetic parameters, except Km for pullulan
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.36
panose
Aspergillus niger
-
mutant enzyme W402F
180
panose
Aspergillus niger
-
wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
9.6
-
pH 5, 60C
25.2
-
pH 3.5, 40C, recombinant IPU expressed in Pichia pastoris
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
3.5
-
recombinant enzyme
3.5
-
assay at
3.5
-
deglycosylated recombinant IPU, native IPU has a similar pH-optimum
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4.5 - 5.6
-
active in the range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
35 - 40
-
deglycosylated recombinant IPU, native IPU has a similar optimum temperature
40
-
at pH 3.5
40
-
F2 at pH 3.5
40
-
assay at
40 - 45
-
recombinant enzyme
45
-
F1 at pH 3.5
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
59000
-
F1 and F2 deglycosylated
136638
59000
-
-
136639
69000
-
F2, SDS-PAGE
136638
71000
-
F1, SDS-PAGE
136638
74000
-
gel filtration
136633
91000
-
recombinant enzyme, SDS-PAGE
136639
200000
-
gel filtration
655376
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
?
-
x * 59000, deglycosylated recombinant IPU, SDS-PAGE
dimer
-
2 * 95000, SDS-PAGE
dimer
-
2 * 95000, SDS-PAGE
-
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
glycoprotein
highly N-glycosylated enzyme with 15 potential N-glycosylation sites (Asn-X-Ser/Thr), the side chain of a glycosylated asparagine residue is important for the stability of the enzyme, enzyme deglycosylation results in a decrease in thermostability. Enzyme deglycosylated with peptide-N-glycosidase F shows virtually no enzymatic activity, while a single GlcNAc residue remains following digestion with Endo H, and the activity of the Endo H-treated wild-type enzyme is 65% compared to intact, non-deglycosylated, wild-type enzyme
glycoprotein
Aspergillus brasiliensis ATCC 9642
-
highly N-glycosylated enzyme with 15 potential N-glycosylation sites (Asn-X-Ser/Thr), the side chain of a glycosylated asparagine residue is important for the stability of the enzyme, enzyme deglycosylation results in a decrease in thermostability. Enzyme deglycosylated with peptide-N-glycosidase F shows virtually no enzymatic activity, while a single GlcNAc residue remains following digestion with Endo H, and the activity of the Endo H-treated wild-type enzyme is 65% compared to intact, non-deglycosylated, wild-type enzyme
-
glycoprotein
-
-
glycoprotein
-
N-glycosylated
glycoprotein
-
recombinant IPU expressed in Pichia pastoris GS115 has a higher carbohydrate content, 41%, than native IPU, 12-15%, or recombinant IPU expressed in Aspergillus oryzae M-2-3, 34%
glycoprotein
-
the sugar content of native IPU is 12.4-15.3%, that of recombinant IPU is 33.8%, deglycosylated enzyme by endoglycosidase H with a sugar content of 2.1% retains 65% of its original activity, kinetics of deglycosylated IPU, hybrid- and/or high-mannose types
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified deglycosylated recombinant enzyme mutant N448A complexed with substrate isopanose, hanging drop vapour diffusion method, 20C, mixing of 0.001 ml protein in 10 mM sodium acetate, pH 3.5, with 0.001 ml of reservoir solution containing 10-13% w/w PEG 8000, and 50 mM sodium acetate, pH 4.5-5.0, cryoprotection with 20% v/v glycerol in reservoir solution, X-ray diffraction structure determination and analysis at 2.2 A resolution, molecular replacement method, the crystal belongs to the P21 space group and contains four molecules in the asymmetric unit, substrate binding structure, overview
for crystallization, the enzyme is treated with endoglycosidase Hf, by hanging drop vapor-diffusion method, unliganded and isopanose-complexed forms of IPU, both solved at 1.7 A resolution. Unliganded IPU belongs to space group P212121, which contains two molecules and 1273 water molecules in an asymmetric unit. IPU is composed of domains N and C joined by a short linker, with electron density maps for 11 or 12 N-acetylglucosamine residues per molecule. Domain N consists of 13 beta-strands and forms a beta-sandwich. Domain C, where the active site is located, forms a right-handed beta-helix. Overall conformation of IPU in complex with isopanose is essentially identical with that of unliganded IPU
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2 - 7
-
at 4C 12 h
136638
2 - 8
-
5C, 24 h
136633
2.4 - 7
-
recombinant IPU expressed in Pichia pastoris, at 4C, retains pullulan-hydrolyzing activity for 12 hours
655036
3 - 7
-
recombinant enzyme, at 4C 12 h
136639
3 - 7
-
deglycosylated recombinant IPU, stable
655805
4 - 7
-
-
136636
5
-
retains activity below pH 5
655376
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
35
-
F2, below, 30 min at pH 3.5
136638
40
-
deglycosylated recombinant IPU, stable up to 40C, native IPU has a similar thermal stability
655805
45
-
F1, below, 30 min at pH 3.5
136638
46.6
50% activity remaining for deglycosylated mutant N448A and for mutant Y440A
732013
49.3
50% activity remaining for mutant N448A
732013
50
-
30 min at pH 3.7-4.5
136633
50
-
below completely stable
136635
52
50% activity remaining for deglycosylated mutant S450A
732013
53
50% activity remaining for the deglycosylated wild-type enzyme
732013
54
50% activity remaining for mutant S450A
732013
55
-
F2, loss of activity
136638
55
50% activity remaining for the wild-type enzyme
732013
60
-
F1, loss of activity
136638
60
-
30 min, stable up to
586944
60
-
60 min, 16% loss of activity
655376
70
-
10 min, 80% loss of activity
136635
70
-
60 min, 30% loss of activity
655376
80
-
inactivated
136635
80
-
pH 5, half-life: 48 min
655376
90
-
pH 5, half-life: 20 min
655376
additional information
-
calcium does not increase the thermostability
655376
OXIDATION STABILITY
ORGANISM
UNIPROT
LITERATURE
photooxidation in presence of Rose Bengal
-
136635
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
4C, no major loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinnat wild-type and mutant enzymes from Pichia pastoris strain GS115 by hydrophobic interaction chromatography
recombinant IPU expressed in Pichia pastoris
-
wild-type and mutant enzymes
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of wild-type and mutant enzymes in Pichia pastoris strain GS115
ipuA gene, expression in Aspergillus oryzae M-2-3
-
ipuA gene, expression in Pichia pastoris GS115
-
recombinant IPU produced by Pichia pastoris
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
N448A
site-directed mutagenesis, glycan-deficient variant, Asn448 is not N-glycosylated, the mutant shows reduced thermal stability compared to the wild-type enzyme
S450A
site-directed mutagenesis, glycan-deficient variant, by replacing Ser450 with Ala, Asn448 is not N-glycosylated, but the Asn448 side chain remains intact, unlike the Asn448Ala mutation, the mutant shows reduced thermal stability compared to the wild-type enzyme
Y440A
site-directed mutagenesis, the mutant shows reduced thermal stability compared to the wild-type enzyme
N448A
Aspergillus brasiliensis ATCC 9642
-
site-directed mutagenesis, glycan-deficient variant, Asn448 is not N-glycosylated, the mutant shows reduced thermal stability compared to the wild-type enzyme
-
S450A
Aspergillus brasiliensis ATCC 9642
-
site-directed mutagenesis, glycan-deficient variant, by replacing Ser450 with Ala, Asn448 is not N-glycosylated, but the Asn448 side chain remains intact, unlike the Asn448Ala mutation, the mutant shows reduced thermal stability compared to the wild-type enzyme
-
Y440A
Aspergillus brasiliensis ATCC 9642
-
site-directed mutagenesis, the mutant shows reduced thermal stability compared to the wild-type enzyme
-
D353N
-
enzyme shows no activity with pullulan and panose as substrate
D372N
-
enzyme shows no activity with pullulan and panose as substrate
D373N
-
enzyme shows no activity with pullulan and panose as substrate
E273N
-
45% of wild-type activity with pullulan as substrate, 74% of wild-type activity with panose as substrate
E356Q
-
38% of wild-type activity with pullulan as substrate, 50% of wild-type activity with panose as substrate
W240F
-
130% of wild-type activity with pullulan as substrate, 160% of wild-type activity with panose as substrate
W31F
-
38% of wild-type activity with pullulan as substrate, 140% of wild-type activity with panose as substrate
W32F
-
90% of wild-type activity with pullulan as substrate, 120% of wild-type activity with panose as substrate
W402F
-
0.4% of wild-type activity with pullulan as substrate, 0.1% of wild-type activity with panose as substrate