Information on EC 2.4.1.230 - kojibiose phosphorylase

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

EC NUMBER
COMMENTARY hide
2.4.1.230
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RECOMMENDED NAME
GeneOntology No.
kojibiose phosphorylase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
2-alpha-D-glucosyl-D-glucose + phosphate = D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
the enzyme from Thermoanaerobacter brockii can act with alpha-1,2-oligoglucans, such as selaginose, as substrate, but more slowly. The enzyme is inactive when dissaccharides with linkages other than alpha-1,2 linkages, such as sophorose, trehalose, neotrehalose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, sucrose and lactose, are used as substrates
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexosyl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
kojibiose degradation
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SYSTEMATIC NAME
IUBMB Comments
2-alpha-D-glucosyl-D-glucose:phosphate beta-D-glucosyltransferase
The enzyme from Thermoanaerobacter brockii can act with alpha-1,2-oligoglucans, such as selaginose, as substrate, but more slowly. The enzyme is inactive when dissaccharides with linkages other than alpha-1,2 linkages, such as sophorose, trehalose, neotrehalose, nigerose, laminaribiose, maltose, cellobiose, isomaltose, gentiobiose, sucrose and lactose, are used as substrates.
CAS REGISTRY NUMBER
COMMENTARY hide
206566-36-1
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
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
2-alpha-D-glucosyl-D-glucose + phosphate
D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
3-O-alpha-D-glucopyranosyl-D-glucopyranose + phosphate
D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
A4XP2
low activity, reaction of nigerose phosphorylase, EC 2.4.1.279
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-
?
3-O-alpha-D-glucosyl-D-glucose + phosphate
D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
alpha,alpha-trehalose + phosphate
D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
A4XP2
very low activity, reaction of trehalose phosphorylase, EC 2.4.1.64
-
-
?
beta-D-glucose 1-phosphate + D-glucose
2-alpha-D-glucosyl-D-glucose + phosphate
show the reaction diagram
-
-
-
-
?
beta-D-glucose 1-phosphate + glycerol
O-alpha-D-glucopyranosyl-(1,1)-glycerol + O-alpha-glucopyranosyl-(1,2)-O-alpha-D-glucopyranosyl-(1,1)-glycerol + phosphate
show the reaction diagram
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0.27% of the activity with D-glucose
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-
?
beta-D-glucose 1-phosphate + myo-inositol
O-alpha-D-glucopyranosyl-(1,1)-myo-inositol + O-alpha-D-glucopyranosyl-(1,5)-myo-inositol + O-alpha-D-glucopyranosyl-(1,2)-O-alpha-D-glucopyranosyl-(1,1)-myo-inositol + O-alpha-D-glucopyranosyl-(1,2)-O-alpha-D-glucopyranosyl-(1,1)-myo-inositol
show the reaction diagram
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6.2% of the activity with D-glucose
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-
?
beta-D-glucose 1-phosphate + raffinose
2-alpha-D-glucopyranosyl-raffinose + 2G(2-alpha-D-glucopyranosyl)2-raffinose + 2G(2-alpha-D-glucopyranosyl)3-raffinose
show the reaction diagram
-
-
-
-
?
beta-D-glucose 1-phosphate + stachyose
2-alpha-D-glucopyranosyl-stachyose + 2G(2-alpha-D-glucopyranosyl)2-stachyose + 2G(2-alpha-D-glucopyranosyl)3-stachyose
show the reaction diagram
-
-
-
-
?
cyclo-[(-6)-alpha-D-Glcp-(1-3)-alpha-D-Glcp-(1-6)-alpha-D-Glcp-(1-3)-alpha-D-Glcp-(1-)] + beta-D-glucose 1-phosphate
cyclo-[(-6)-alpha-D-Glcp-(1-3)-alpha-D-Glcp-(1-6)-[alpha-D-Glcp-(1-2)]-alpha-D-Glcp-(1-3)-alpha-D-Glcp-(1-)] + phosphate
show the reaction diagram
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i.e. CTS
major product, i.e. 2-O-alpha-D-glucopyranosyl-CTS
-
?
D-glucose + beta-D-glucose 1-phosphate
2-alpha-D-glucosyl-D-glucose + phosphate
show the reaction diagram
D-glucose + O-beta-D-fructofuranosyl-(2-1)-O-beta-D-fructofuranosyl-(2-1)-O-beta-D-fructofuranosyl-(2-1)-glucopyranoside
[O-alpha-D-glucopyranosyl-1(1-2)]n-O-[beta-D-fructofuranosyl-(2-1)]3-alpha-D-glucopyranoside + phosphate
show the reaction diagram
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n = 1 or 2
?
kojibiose + phosphate
beta-D-glucose-1-phosphate + D-glucose
show the reaction diagram
kojitetraose + phosphate
beta-D-glucose 1-phosphate + kojitriose
show the reaction diagram
kojitriose + phosphate
beta-D-glucose 1-phosphate + 2-alpha-D-glucosyl-D-glucose
show the reaction diagram
maltose + phosphate
D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
A4XP2
very low, activity, reaction of maltose phosphorylase, EC 2.4.1.8
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-
?
O-alpha-D-xylopyranosyl-(1-2)-beta-D-fructofuranoside + beta-D-glucose 1-phosphate
O-alpha-D-glucopyranosyl-(1-[2-O-alpha-D-glucopyranosyl-1]0-2)-O-alpha-D-xylopyranosyl-(1-2)-beta-D-fructofuranoside + phosphate
show the reaction diagram
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r
O-alpha-D-xylopyranosyl-(1-2)-beta-D-fructofuranoside + beta-D-glucose 1-phosphate
O-alpha-D-glucopyranosyl-(1-[2-O-alpha-D-glucopyranosyl-1]1-2)-O-alpha-D-xylopyranosyl-(1-2)-beta-D-fructofuranoside + phosphate
show the reaction diagram
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r
O-alpha-D-xylopyranosyl-(1-2)-beta-D-fructofuranoside + beta-D-glucose 1-phosphate
O-alpha-D-glucopyranosyl-(1-[2-O-alpha-D-glucopyranosyl-1]2-2)-O-alpha-D-xylopyranosyl-(1-2)-beta-D-fructofuranoside + phosphate
show the reaction diagram
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r
palatinose + beta-D-glucose 1-phosphate
2G(2-alpha-D-glucopyranosyl)2-palatinose + phosphate
show the reaction diagram
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r
palatinose + beta-D-glucose 1-phosphate
2G-alpha-D-glucopyranosyl-palatinose + phosphate
show the reaction diagram
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r
palatinose + beta-D-glucose 1-phosphate
O-alpha-D-glucopyranosyl-(1-2)-O-alpha-D-glucopyranosyl-(1-2)-O-alpha-D-glucopyranosyl-(1-6)-D-fructofuranose + phosphate
show the reaction diagram
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r
palatinose + beta-D-glucose 1-phosphate
O-alpha-D-glucopyranosyl-(1-2)-O-alpha-D-glucopyranosyl-(1-6)-D-fructofuranose + phosphate
show the reaction diagram
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r
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
2-alpha-D-glucosyl-D-glucose + phosphate
D-glucose + beta-D-glucose 1-phosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
CdCl2
1 mM, elevates hydrolysis activity by 33%
CuCl2
1 mM, elevates hydrolysis activity by 40%
ZnCl2
1 mM, elevates hydrolysis activity by 36%
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
MnCl2
1 mM, 34% loss of activity
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.5 - 84
2-alpha-D-glucosyl-D-glucose
3.84 - 25.4
beta-D-glucose
0.77 - 8.57
beta-D-glucose 1-phosphate
36.8
D-glucose
pH 6.0, 90C
0.77 - 5.21
kojibiose
0.75 - 1.34
phosphate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.097 - 37.7
2-alpha-D-glucosyl-D-glucose
37.7
phosphate
Pyrococcus sp.
I3RFR4
pH 6.0, 90C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011 - 14.9
2-alpha-D-glucosyl-D-glucose
5735
28.1
phosphate
Pyrococcus sp.
I3RFR4
pH 6.0, 90C
16
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10.7
pH 6.0, 80C, substrate: kojibiose
31.1
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purified recombinant enzyme, pH 6.0, 50C, phosphorolysis
191.3
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purified recombinant enzyme, pH 6.0, 50C, synthesis
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6
over 90% of maximal activity at pH 4.0 and at pH 6.0
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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phosphorolysis and reverse phosphorolysis
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
67.5 - 70
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mutant D513N
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.3 - 4.4
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isoelectric focusing
PDB
SCOP
CATH
ORGANISM
UNIPROT
Caldicellulosiruptor saccharolyticus (strain ATCC 43494 / DSM 8903 / Tp8T 6331)
Caldicellulosiruptor saccharolyticus (strain ATCC 43494 / DSM 8903 / Tp8T 6331)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
170000
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recombinant enzyme, gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified enzyme in complex with glucose and phosphate and in complex with kojibiose and sulfate, Glc-PO4 crystals are obtained at 25C by sitting drop vapor diffusion method, mixing 0.001 ml of 5.9 m/ml protein in 5 mM Tris/HCl, pH 7.5, with an equal volume of reservoir solution containing 5 mM D-glucose, 5 mM sodium phosphate, pH 8.5, 10% v/v 2-propanol, 10% w/v PEG 3350, and 0.1 M Tris/HCl, pH 8.5, the kojibiose-SO4 crystals are obtained at 25C by hanging drop vapor diffusion method, mixing of 0.001 ml of 5.4 mg/ml protein solution in 5 mM Tris/HCl, pH 7.5, with an equal volume of reservoir solution containing 5 mM kojibiose, 0.2 M NaCl, 2.0 M (NH4)2SO4, and 0.1 M sodium cacodylate, pH 6.5, X-ray diffraction structure determination and analysis at 2.05-2.80 A resolution, structures PDB IDs 3WIR and 3WIQ, molecular replacement with the maltose phosphorylase structure from Lactobacillus brevis LbMP, PDB ID 1H54, as the search model
A4XP2
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.5 - 10
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recombinant enzyme, stable at, phosphorolysis
721869
5.5 - 10.5
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recombinant enzyme, stable at, synthesis
721869
5.5 - 9.7
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stable
644894
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
60
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50% residual activity, mutant D513N 135 h, wild-type 85 h
85
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recombinant enzyme, stable up to, phosphorolysis
95
half-life: 71 h
100
2 h, over 40% of the activity remains. Half-life: 1.9 h
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
chimeric enzymes
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kojibiose phosphorylase is purified from a cell-free extract of Thermoanaerobacter brockii
recombinant enzyme
recombinant His-tagged LaMP loop 3 mutants from Escherichia coli strain BL21(DE3) by affinity chromatography and dialysis
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recombinant wild-type and mutant enzymes
A4XP2
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DNA and amino acid sequence determination and analysis, expression in Escherichia coli
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expression of His-tagged LaMP loop 3 mutants in Escherichia coli strain BL21(DE3)
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heterologous expression in Escherichia coli
kojibiose phosphorylase gene and trehalose phosphorylase gene are intracellularly hyperexpressed under the control of the Bacillus amyloliquefaciens alpha-amylase promoter in Bacillus subtilis
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recombinant expression of wild-type and mutant enzymes
A4XP2
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E392R
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E392R/T417A
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E392R/T417I
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
T417A
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
T417F
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
T417I
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W391M/E392V
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W391M/E392V/T417A
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W391M/E392V/T417I
A4XP2
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
D177N
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106% of wild-type activity
D271N
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69% of wild-type activity
D340N
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89% of wild-type activity
D362N
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complete loss of enzymic activity
D459N
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increase in Km-values for kojibiose, beta-D-glucose 1-phosphate and glucose
D513N
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enhanced thermostablility and thermoactivity
E284Q
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103% of wild-type activity
E642Q
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complete loss of enzymic activity
K114Q
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85% of wild-type activity
K403Q
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73% of wild-type activity
K614Q
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complete loss of enzymic activity
K749Q
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78% of wild-type activity
R137Q
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61% of wild-type activity
R33Q
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31% of wild-type activity
R476Q
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50% of wild-type activity
R48Q
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62% of wild-type activity
D177N
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106% of wild-type activity
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D362N
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complete loss of enzymic activity
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E642Q
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complete loss of enzymic activity
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K403Q
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73% of wild-type activity
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K614Q
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complete loss of enzymic activity
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis
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