Information on EC 2.4.1.230 - kojibiose phosphorylase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, 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
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-
?
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
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-
-
-
?
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, 90°C
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
pH 6.0, 90°C
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0011 - 14.9
2-alpha-D-glucosyl-D-glucose
28.1
phosphate
pH 6.0, 90°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10.7
pH 6.0, 80°C, substrate: kojibiose
31.1
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purified recombinant enzyme, pH 6.0, 50°C, phosphorolysis
191.3
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purified recombinant enzyme, pH 6.0, 50°C, 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
UNIPROT
ORGANISM
Caldicellulosiruptor saccharolyticus (strain ATCC 43494 / DSM 8903 / Tp8T 6331);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
80000
x * 80000, SDS-PAGE
81000
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2 * 81000, recombinant enzyme, SDS-PAGE
83000
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x * 83000, SDS-PAGE
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 25°C 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 25°C 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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