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 ACCESSION NO.
LITERATURE
hydrolysis of pullulan to isopanose (6-alpha-maltosylglucose)
show the reaction diagram
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
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 ACCESSION NO.
COMMENTARY
LITERATURE
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
SEQUENCE CODE
SEQUENCE DB
SOURCE
T6, single enzyme with G2-dextranase EC 3.2.1.94 and isopullanase activity
-
-
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
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
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
-
-
-
-
?
panose + H2O
?
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
-
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
-
-
-
-
-
pullulan + H2O
isopanose + ?
show the reaction diagram
O00105
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 ACCESSION NO.
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 ACCESSION NO.
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]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
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]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.36
-
panose
-
mutant enzyme W402F
180
-
panose
-
wild-type enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
9.6
-
-
pH 5, 60C
25.2
-
-
pH 3.5, 40C, recombinant IPU expressed in Pichia pastoris
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
3.5
-
-
recombinant enzyme
3.5
-
-
assay at
3.5
-
-
deglycosylated recombinant IPU, native IPU has a similar pH-optimum
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
4.5
5.6
-
active in the range
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
35
40
-
deglycosylated recombinant IPU, native IPU has a similar optimum temperature
40
45
-
recombinant enzyme
40
-
-
at pH 3.5
40
-
-
F2 at pH 3.5
40
-
-
assay at
45
-
-
F1 at pH 3.5
60
-
-
-
LOCALIZATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
59000
-
-
F1 and F2 deglycosylated
69000
-
-
F2, SDS-PAGE
71000
-
-
F1, SDS-PAGE
74000
-
-
gel filtration
91000
-
-
recombinant enzyme, SDS-PAGE
200000
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
?
-
x * 59000, deglycosylated recombinant IPU, SDS-PAGE
dimer
-
2 * 95000, SDS-PAGE
dimer
-
2 * 95000, SDS-PAGE
-
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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 ACCESSION NO.
LITERATURE
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
O00105
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
2
7
-
at 4C 12 h
2
8
-
5C, 24 h
2.4
7
-
recombinant IPU expressed in Pichia pastoris, at 4C, retains pullulan-hydrolyzing activity for 12 hours
3
7
-
recombinant enzyme, at 4C 12 h
3
7
-
deglycosylated recombinant IPU, stable
5
-
-
retains activity below pH 5
TEMPERATURE STABILITY
TEMPERATURE STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
35
-
-
F2, below, 30 min at pH 3.5
40
-
-
deglycosylated recombinant IPU, stable up to 40C, native IPU has a similar thermal stability
45
-
-
F1, below, 30 min at pH 3.5
50
-
-
30 min at pH 3.7-4.5
50
-
-
below completely stable
55
-
-
F2, loss of activity
60
-
-
F1, loss of activity
60
-
-
30 min, stable up to
60
-
-
60 min, 16% loss of activity
70
-
-
10 min, 80% loss of activity
70
-
-
60 min, 30% loss of activity
80
-
-
inactivated
80
-
-
pH 5, half-life: 48 min
90
-
-
pH 5, half-life: 20 min
additional information
-
-
calcium does not increase the thermostability
OXIDATION STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
photooxidation in presence of Rose Bengal
-
136635
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
4C, no major loss of activity
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
recombinant IPU expressed in Pichia pastoris
-
wild-type and mutant enzymes
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
ipuA gene, expression in Aspergillus oryzae M-2-3
-
ipuA gene, expression in Pichia pastoris GS115
-
recombinant IPU produced by Pichia pastoris
O00105
ENGINEERING
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
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