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Enzyme Nomenclature
Information on EC 2.4.1.20 - cellobiose phosphorylase
Word Map on EC 2.4.1.20
- 2.4.1.20
-
phosphorolysis
-
gilvus
-
cellvibrio
-
phosphorylases
-
cellodextrins
- thermocellum
-
cellulomonas
-
cellulolytic
- synthesis
-
ruminococcus
- alpha-d-glucose
- laminaribiose
- alpha-d-glucose-1-phosphate
-
phosphorolyzed
-
cellobiose-grown
- d-glucosamine
- 6-deoxy-d-glucose
- cellotriose
- fibrobacter
- beta-d-glucose
- analysis
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The enzyme appears in viruses and cellular organisms
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EC NUMBER 
COMMENTARY 
2.4.1.20
-
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RECOMMENDED NAME
GeneOntology No.
cellobiose phosphorylase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cellobiose + phosphate = alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
cellobiose + phosphate = alpha-D-glucose 1-phosphate + D-glucose
computational analyses to elucidate the conformational itinerary along the reaction pathway of this enzyme using the substrate binding structure determined from crystal structure, predicted reaction pathway, overview
-
cellobiose + phosphate = alpha-D-glucose 1-phosphate + D-glucose
reaction proceeds through a random-ordered bi-bi mechanism, by which phosphate and cellobiose bind in random order and D-glucose is released before glucose 1-phosphate
-
cellobiose + phosphate = alpha-D-glucose 1-phosphate + D-glucose
reaction proceeds through a random-ordered bi-bi mechanism, by which phosphate and cellobiose bind in random order and D-glucose is released before glucose 1-phosphate
-
-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hexosyl group transfer
-
-
-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
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SYSTEMATIC NAME
IUBMB Comments
cellobiose:phosphate alpha-D-glucosyltransferase
-
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CAS REGISTRY NUMBER
COMMENTARY
9030-20-0
-
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
489388, 637858, 637859, 637860, 637861, 637862, 637867, 657459, 671874, 672931, 691642, 705837, 736733
-
-
strain YM4 (AB013109). To convert cellobiose into amylose: the cellobiose phosphorylase catalyzes phosphorolysis of cellobiose into G1-P and glucose, then alpha-glucan phosphorylase catalyses the polymerization of alpha-1,4-glucan, using G1-P as a carbohydrate donor and maltotetraose as the initial carbohydrate acceptor. Thus, glucosyl moieties of cellobiose molecules are successively transferred to the non-reducing ends of glucan to elongate the chain. The optimal conditions are: thirty milligrams per milliliters (87.7 mM) cellobiose, 30 mM sodium phosphate (pH 7.0), 30 microg/ml cellobiose phosphorylase, 30 microg/ml alpha-glucan phosphorylase, and 75 microM maltotetraose are incubated at 45°C
-
-
strain YM4 (AB013109). To convert cellobiose into amylose: the cellobiose phosphorylase catalyzes phosphorolysis of cellobiose into G1-P and glucose, then alpha-glucan phosphorylase catalyses the polymerization of alpha-1,4-glucan, using G1-P as a carbohydrate donor and maltotetraose as the initial carbohydrate acceptor. Thus, glucosyl moieties of cellobiose molecules are successively transferred to the non-reducing ends of glucan to elongate the chain. The optimal conditions are: thirty milligrams per milliliters (87.7 mM) cellobiose, 30 mM sodium phosphate (pH 7.0), 30 microg/ml cellobiose phosphorylase, 30 microg/ml alpha-glucan phosphorylase, and 75 microM maltotetraose are incubated at 45°C
-
-
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
alpha-D -glucose 1-fluoride + D-glucose
cellobiose + fluoride
-
-
-
-
ir
alpha-D-glucose 1-phosphate + 5a-carba-alpha-D,L-glucopyranose
4-O-beta-D-glucopyranosyl-5a-carba-beta-D-glucopyranose + phosphate
-
very poor substrate
-
-
?
alpha-D-glucose 1-phosphate + 5a-carba-beta-D,L-glucopyranose
?
-
much better substrate than alpha-anomer, 22% of activity with D-glucose
-
-
?
alpha-D-glucose 1-phosphate + butyl beta-D-glucoside
? + phosphate
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + cellobiose
? + phosphate
-
no substrate for wilalpha-D-type
-
-
r
alpha-D-glucose 1-phosphate + D-glucosamine
4-O-beta-D-glucopyranosyl-D-glucosamine
-
-
-
-
?
alpha-D-glucose 1-phosphate + D-mannose
4-O-beta-D-glucopyranosyl-D-mannose
-
-
-
-
?
alpha-D-glucose 1-phosphate + ethyl beta-D-glucoside
? + phosphate
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + methyl alpha-D-glucoside
? + phosphate
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + methyl beta-D-glucoside
? + phosphate
-
no substrate for wild-type
-
-
r
alpha-D-glucose 1-phosphate + N-acetyl-D-glucosamine
4-O-beta-D-glucopyranosyl-N-acetyl-D-glucosamine
-
substrate for mutant Y648V only
-
-
?
alpha-D-glucose 1-phosphate + phenyl beta-D-glucoside
? + phosphate
-
no substrate for wild-type
-
-
r
beta-D-glucose 1-phosphate + alpha-D-glucopyranosyl fluoride
beta-cellobiose + HF
-
beta-D-glucopyranosyl fluoride is not ulilized
-
?
cellobiose + arsenate
D-glucose + alpha-D-glucose 1-arsenate
-
-
alpha-D-glucose 1-arsenate hydrolyses spontaneously to form D-glucose and arsenate
ir
cellobiose + phosphate
D-glucose 1-phosphate + D-glucose
-
-
-
-
?
D-glucal + 2-deoxy-2-(hydroxymethyl)-beta-D-xylopyranose
2-deoxy-3-O-(2-deoxy-beta-D-arabino-hexopyranosyl)-2-(hydroxymethyl)-beta-D-xylopyranose + ?
-
-
-
-
r
D-glucal + D-glucose
2-deoxy-beta-D-arabino-hexopyranosyl-(1->4)-D-glucose + ?
-
-
-
-
r
D-glucal + D-mannose
2-deoxy-beta-D-arabino-hexopyranosyl-(1->4)-D-mannose + ?
-
-
-
-
r
D-glucal + D-xylose
2-deoxy-beta-D-arabino-hexopyranosyl-(1->4)-D-xylose + ?
-
-
-
-
r
D-glucal + phosphate
2-deoxy-alpha-D-glucose 1-phosphate
-
D-glucal is a slow alternative donor substrate for stereospecific glycosyl transfer to phosphate. Reaction follows a stereochemical course where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism involves direct protonation of D-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered D-glucose next to D-glucal and phosphate, the enzyme produces 2-deoxy-beta-D-glucosyl-(1->4)-D-glucose and 2-deoxy-alpha-D-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-beta-D-glucosyl-(1->4)-D-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of enzyme, D-glucal, phosphate,and D-glucose
-
-
?
lactose + phosphate
alpha-D-galactose 1-phosphate + D-glucose
-
-
-
r
alpha-D-glucose 1-phosphate + 1,5-anhydro-D-glucitol
? + phosphate
-
-
-
-
r
alpha-D-glucose 1-phosphate + 1,5-anhydro-D-glucitol
? + phosphate
-
-
-
-
r
alpha-D-glucose 1-phosphate + 2-deoxy-D-glucose
?
-
-
-
-
?
alpha-D-glucose 1-phosphate + 2-deoxy-D-glucose
?
-
-
-
-
?
alpha-D-glucose 1-phosphate + 2-deoxy-D-glucose
? + phosphate
-
-
-
-
r
alpha-D-glucose 1-phosphate + 6-deoxy-D-glucose
?
-
much better substrate than 2-deoxy-D-glucose
-
-
?
alpha-D-glucose 1-phosphate + 6-deoxy-D-glucose
?
-
-
-
-
?
alpha-D-glucose 1-phosphate + 6-deoxy-D-glucose
?
-
-
-
-
?
alpha-D-glucose 1-phosphate + 6-deoxy-D-glucose
? + phosphate
-
-
-
-
r
alpha-D-glucose 1-phosphate + D-arabinose
?
-
very poor substrate
-
-
?
alpha-D-glucose 1-phosphate + D-glucosamine
?
-
-
-
-
?
alpha-D-glucose 1-phosphate + D-glucose
cellobiose + phosphate
-
in synthesis reaction, glucose is best substrate
-
-
r
alpha-D-glucose 1-phosphate + D-glucose
cellobiose + phosphate
-
in synthesis reaction, glucose is best substrate
-
-
r
alpha-D-glucose 1-phosphate + D-mannose
?
-
-
-
-
?
alpha-D-glucose 1-phosphate + D-xylose
?
-
no activity with L-isomer
-
-
?
alpha-D-glucose 1-phosphate + D-xylose
?
-
no activity with L-isomer
-
-
?
alpha-D-glucose 1-phosphate + D-xylose
?
-
higher specific activity than with D-glucose as acceptor
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
beta-D-cellobiose is phosphorylyzed faster than alpha-anomer, ordered bi-bi mechanism
beta-D-glucose was shown to be the 4 times better acceptor compared to alpha-D-glucose in the reverse reaction
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
sequential bi-bi mechanism
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
CBP catalyzes the phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of anomeric configuration, computational docking, overview
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
beta-cellobiose preferred over alpha-anomer
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
ternary-complex kinetic mechanism
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
important for energy efficient catabolism of cellobiose in cytoplasm
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
ordered bi-bi mechanism
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
beta-cellobiose preferred over alpha-anomer
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
the enzyme shows beta-anomer selective glucosylation activity towards disaccharides
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
ordered bi-bi mechanism
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
beta-cellobiose preferred over alpha-anomer
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
the enzyme shows beta-anomer selective glucosylation activity towards disaccharides
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
random ordered bi-bi mechanism
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
sequential bi-bi mechanism
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
O52504
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
O52504
nonconventional pathway for glucan utilization
-
-
-
additional information
?
-
only disaccharide accepted as substrate
-
-
-
additional information
?
-
only disaccharide accepted as substrate
-
-
-
additional information
?
-
beta-glucosylation of alkyls by the enzyme using either cellobiose or alpha-D-glucose 1-phosphate as donors, active with methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, n-pentanol, n-hexanol, n-heptanol, 1,2-butanediol, 1,3-butanediol, and 1,4-butanediol
-
-
-
additional information
?
-
beta-glucosylation of alkyls by the enzyme using either cellobiose or alpha-D-glucose 1-phosphate as donors, active with methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, tert-butanol, n-pentanol, n-hexanol, n-heptanol, 1,2-butanediol, 1,3-butanediol, and 1,4-butanediol
-
-
-
additional information
?
-
-
no activity with D-xylose and D-mannose
-
-
-
additional information
?
-
-
no activity detected with glucose-acceptor different from D-glucose
-
-
-
additional information
?
-
O52504
only disaccharide accepted as substrate
-
-
-
additional information
?
-
-
only disaccharide accepted as substrate
-
-
-
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
Q7WTR6
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
important for energy efficient catabolism of cellobiose in cytoplasm
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
Q59316
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
Q59316
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
Q8VP44
the enzyme shows beta-anomer selective glucosylation activity towards disaccharides
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
Q8VP44
the enzyme shows beta-anomer selective glucosylation activity towards disaccharides
-
-
r
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
-
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
-
-
-
-
?
cellobiose + phosphate
alpha-D-glucose 1-phosphate + D-glucose
O52504
nonconventional pathway for glucan utilization
-
-
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
additional information
-
no influence of metal ions on reaction
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INHIBITORS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
D-glucose
-
mixed type inhibition against cellobiose, pure beta-D-glucose inhibits stronger than mixture of both anomers
D-glucose
-
substrate inhibition, competitive with glucose-1-phosphate
D-glucose
-
inhibition of the synthetic reaction, binds competitively at the glucose 1-phosphate site and uncompetitvely at the ternary enzyme-glucose 1-phosphate-glucose complex
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
8 - 9
1,5-anhydroglucitol
-
enzyme assay at 37°C in 50 mM Tris/HCl (pH 7.0)
33.7
butyl beta-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
1009
methyl alpha-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
31.2
phenyl beta-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
additional information
additional information
-
simulation of molecular dynamics of the enzyme, the 1S3 pre-transition state conformer is highly stable compared with other conformers, and a conformational change from 4C1 to 1,4B occurs
-
3.28
alpha-D-glucose 1-phosphate
-
pH 6.6, 30°C, recombinant His6-tagged enzyme
11.3
alpha-D-glucose 1-phosphate
-
mutant enzyme C485A, at pH 6.0 and 37°C
15.7
alpha-D-glucose 1-phosphate
-
mutant enzyme Y648V, at pH 6.0 and 37°C
30.7
alpha-D-glucose 1-phosphate
-
mutant enzyme Y648F, at pH 6.0 and 37°C
2.9
cellobiose
pH 6.6, 37°C, recombinant mutant T508A/N667T; pH 6.6, 37°C, recombinant wild-type enzyme
3.3
cellobiose
pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S
14.7
cellobiose
-
mutant R48R/Q130H/K131Y/K142R/S411G/A423S/V526A/A781K, pH 7.2, 40°C
15.9
cellobiose
-
mutant Q130H/K131Y/S411G/A423S/A781K, pH 7.2, 40°C
138
ethyl beta-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
-
330
lactose
pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S
79.8
methyl beta-D-glucoside
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
3.5
1,5-anhydroglucitol
Cellulomonas gilvus
-
enzyme assay at 37°C in 50 mM Tris/HCl (pH 7.0)
9
butyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
6
methyl alpha-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
4
phenyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
15.4
phosphate
Cellulomonas uda
-
pH 6.6, 30°C, native enzyme; pH 6.6, 30°C, recombinant His6-tagged enzyme
43.2
alpha-D-glucose 1-phosphate
Cellulomonas uda
-
pH 6.6, 30°C, native enzyme; pH 6.6, 30°C, recombinant His6-tagged enzyme
64.2
alpha-D-glucose 1-phosphate
Ruminococcus albus
-
mutant enzyme Y648F, at pH 6.0 and 37°C
96.5
alpha-D-glucose 1-phosphate
Ruminococcus albus
-
mutant enzyme C485A, at pH 6.0 and 37°C
1.5
cellobiose
Ruminiclostridium thermocellum
-
mutant R48R/Q130H/K131Y/K142R/S411G/A423S/V526A/A781K, pH 7.2, 40°C
2.1
cellobiose
Ruminiclostridium thermocellum
-
mutant Q130H/K131Y/S411G/A423S/A781K, pH 7.2, 40°C
2.9
cellobiose
Ruminiclostridium thermocellum
-
mutant A423S, pH 7.2, 40°C; mutant A781 K, pH 7.2, 40°C
3.28
cellobiose
Cellulomonas uda
Q7WTR6
pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S
29.1
cellobiose
Cellulomonas uda
-
pH 6.6, 30°C, native enzyme; pH 6.6, 30°C, recombinant His6-tagged enzyme
27.5
D-glucose
Cellulomonas uda
-
pH 6.6, 30°C, native enzyme; pH 6.6, 30°C, recombinant His6-tagged enzyme
8.7
ethyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
-
1.11
lactose
Cellulomonas uda
Q7WTR6
pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S
7.9
methyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.266
butyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
20171
0.006
methyl alpha-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
5684
0.0376
N-acetyl-D-glucosamine
Ruminococcus albus
-
mutant enzyme Y648V, at pH 6.0 and 37°C
300
0.129
phenyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
4858
0.0829
alpha-D-glucose 1-phosphate
Ruminococcus albus
-
mutant enzyme Y648V, at pH 6.0 and 37°C
107
2.09
alpha-D-glucose 1-phosphate
Ruminococcus albus
-
mutant enzyme Y648F, at pH 6.0 and 37°C
107
8.55
alpha-D-glucose 1-phosphate
Ruminococcus albus
-
mutant enzyme C485A, at pH 6.0 and 37°C
107
63.2
alpha-D-glucose 1-phosphate
Ruminococcus albus
-
wild type enzyme, at pH 6.0 and 37°C
107
0.103
cellobiose
Ruminiclostridium thermocellum
-
mutant R48R/Q130H/K131Y/K142R/S411G/A423S/V526A/A781K, pH 7.2, 40°C
82
0.134
cellobiose
Ruminiclostridium thermocellum
-
mutant Q130H/K131Y/S411G/A423S/A781K, pH 7.2, 40°C
82
0.99
cellobiose
Cellulomonas uda
Q7WTR6
pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S
82
0.063
ethyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
19675
0.0034
lactose
Cellulomonas uda
Q7WTR6
pH 6.6, 37°C, recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S
114
0.099
methyl beta-D-glucoside
Cellulomonas uda
-
mutant N156D/N163D/T508I/E649G/N667A, pH 6.6, 37°C
3478
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
13
4-thio-cellobiose
-
in presence of saturating concentrations of phosphate
255
D-glucose
-
pH 6.6, 30°C, substrate inhibition, recombinant His6-tagged enzyme
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
0.1
purifed recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S, substrate lactose
1.35
purifed recombinant mutant A397V/T508A/A512T/D557N/N667T/G681S, substrate cellobiose
additional information
additional information
-
the activity on D-glucal is approximately 500times less than that on alpha-D-glucose 1-phosphate at 10 mM of each donor with 10 mM D-glucose by comparing the initial rate of the production
additional information
-
the cellobiose phosphorylase activites in the concentrated cell extract is 84 IU/ml.
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6.2
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pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37
60
80
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TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PDB
SCOP
CATH
ORGANISM
UNIPROT
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
85000
92000
93000
150000
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SUBUNITS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
monomer
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Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
at 25°C using the sitting-drop vapour diffusion method. Cellobiose phosphorylase-SO4 and cellobiose phosphorylase-PO4 structures are determined at 2.0 and 2.1 A resolution to R factor (Rfree) of 17.6% (21.3%) and 18.0% (22%) respectively. The pH values for crystallization are pH 8.2 for cellobiose phosphorylase-PO4 and pH 6.5 for cellobiose phosphorylase-SO4.
-
crystallization of recombinant enzyme by the sitting-drop vapour-diffusion method using 10 mg/ml purified enzyme, 1.5 M ammonium sulfate, 0.1 M MES buffer pH 7.0 and 5 mM glucose. A suitable crystal is obtained after 10 d incubation at 298 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 84.77, b = 98.31, c = 104.04 A, beta = 102.73°. X-ray diffraction data to 2.1 A resolution collected at KEK-PF BL-5A
-
in complex with phosphate, to 2.4 A resolution. Enzyme is composed of four distinct domains, an N-terminal domain with residues 1-279, a helical linker with residues 280-314, an (alpha/beta)6-barrel domain with residues 321-734, and a C-terminal beta-sandwich domain with residues 315-320 and 735-811. The side chains of His653, Gln699 and Thr718 and the backbone N-atom of Gly719 coordinate the phosphate bound in the interior of the (alpha/beta)6-barrel domain. Two short helical extensions, residues 158-169, from the N-terminal domain of the adjacent subunit extend into the cleft which runs along the face of the (alpha/beta)6-barrel domain, forming an active-site pocket. An additional loop, residues 488-507, lays on top of the active-site pocket, forming a restrictive active-site pocket that is large enough to bind a disaccharide but not large enough to bind an oligosaccharide
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
4 - 9
-
most stable at pH 7, rapid loss of activity at different pH
637863
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
37 - 60
-
in contrast to the rapid inactivation at 60°C, the enzyme is remarkably stable at 37°C. At 50°C, the enzyme retains over 58% of its initial activity after 24 h
40
60
70
85
O52504
active for 2 h in presence of substrate, without substrate little activity after 15 min
70
-
half-life of wild-type 8.3 min, mutant Q130H/K131Y/S411G/A423S/A781K 17.7 min, mutant R48R/Q130H/K131Y/K142R/S411G/A423S/V526A/A781K 24.6 min
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GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
enzyme immobilization with cross-linked enzyme aggregates boosts the half-life at 50°C from 34 h to almost 11 days
-
storage of purified enzyme at -20°C without thiol reagents causes rapid loss of activity
-
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Ethanol
Ethyl acetate
Methanol
Ethanol
-
ethanol reduces the time required to reduce the initial enzymatic activity by 50% at 50°C from over 32 h to less than 1 h
Ethanol
-
ethanol reduces the time required to reduce the initial enzymatic activity by 50% at 50°C from over 32 h to less than 1 h
-
Ethyl acetate
-
the enzyme displays a relative activity of 92% in 37.5% (v/v) ethyl acetate
Ethyl acetate
-
the enzyme displays a relative activity of 92% in 37.5% (v/v) ethyl acetate
-
Methanol
-
methanol reduces the time required to reduce the initial enzymatic activity by 50% at 50°C from over 32 h to less than 4 h
Methanol
-
methanol reduces the time required to reduce the initial enzymatic activity by 50% at 50°C from over 32 h to less than 4 h
-
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Ni-NTA agarose slurry, Superdex 200 column and Mono Q column
-
recombinant enzyme from E. coli
recombinant enzyme with a cellobiose-binding intein tag, development of a simple, low-cost, and scalable protein purification method using a biodegradable regenerated amorphous cellulose, affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage, method evaluation, overview
-
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli by immobilized metal ion affinity chromatography
use of thiol reagent necessary during purification to maintain activity
-
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CP gene, expression of His-tagged wild-type and mutant enzymes in Escherichia coli
expressed in Escherichia coli
expressed in Escherichia coli, part of oligosaccharide catabolic cluster
O52504
expressed in Saccharomyces cerevisiae strain D452-2
expressed in Saccharomyces cerevisiae strain D452-2 and Escherichia coli BL21(DE3)
-
expression in Escherichia coli
expression of a functional recombinant enzyme containing an N-terminal metal affinity fusion peptide in Escherichia coli. Catalytic properties of the natural enzyme are retained completely in the recombinant cellobiose phosphorylase
-
expression of intein-tagged enzyme in Escherichia coli strain BL21, subcloning in Escherichia coli strain DH5alpha
-
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
A397V
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows reduced activity with lactose compared to the wild-type enzyme
A397V/T508A/A512T/D557N/N667T/G681S
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows 3fold increased activity with lactose and 6fold decreased activity with cellobiose compared to the wild-type enzyme
A512T
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows similar activity with lactose compared to the wild-type enzyme
D557N
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows similar activity with lactose compared to the wild-type enzyme
E649C
-
mutant accepts methyl beta-D-glucoside and ethyl beta-D-glucoside as substrates
G681S
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows reduced activity with lactose compared to the wild-type enzyme
T508A/N667T
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows increased activity with lactose compared to the wild-type enzyme
T508I/N667A
A423S
-
mutation introduced to increase thermostability, mutant displays a half-life of 11.3 min at 70°C
A781K
-
mutation introduced to increase thermostability, mutant displays a half-life of 15.3 min at 70°C
Q130H/K131Y/S411G/A423S/A781K
-
mutations introduced to increase thermostability, mutant displays a half-life of 17.7 min at 70°C
R48R/Q130H/K131Y/K142R/S411G/A423S/V526A/A781K
-
mutations introduced to increase thermostability, mutant displays a half-life of 24.6 min at 70°C
S411G
-
mutation introduced to increase thermostability, mutant displays a half-life of 13.6 min at 70°C
C485A
-
the mutant shows 43% of wild type activity with cellobiose. The mutant shows higher preference for D-glucosamine than the wild type enzyme
Y648F
-
The mutant shows 23% of wild type activity with cellobiose. Apparent kcat/Km values of the mutant for D-mannose and 2-deoxy-D-glucose are 8.2 and 4.0fold higher than those of the wild type enzyme, respectively
Y648V
-
the mutant shows 0.888% of wild type activity with cellobiose and has synthetic activity toward N-acetyl-D-glucosamine
additional information
T508I/N667A
random, site-saturation, and site-directed mutagenesis, the mutant enzyme shows 7.5fold increased activity with lactose compared to the wild-type enzyme
T508I/N667A
-
mutant accepts methyl beta-D-glucoside and ethyl beta-D-glucoside as substrates
additional information
-
random, site-saturation, and site-directed mutagenesis for directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase activity, useful for the production of alpha-D-galactose 1-phosphate, overview. Location of mutations in the 3D structure, overview
additional information
random, site-saturation, and site-directed mutagenesis for directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase activity, useful for the production of alpha-D-galactose 1-phosphate, overview. Location of mutations in the 3D structure, overview
additional information
-
method development using the enzyme for production of D-glucose 1-phosphate, which is an expensive substrate for synthesis of amylose by alpha-glucan phosphorylase, overview
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
analysis
-
quantitative determination of cellobiose in presence of glucose or glucose-1-phosphate
synthesis
synthesis
-
ability of the phosphorylase to utilize alpha-D -glucose 1-fluoride as alternate glucosyl donor in place of alpha-D-glucose 1-phosphate for the synthesis of alpha-1,4-glucosides under thermodynamic control in close to 100% yield
synthesis
-
the cellobiose phosphorylases in the cell extract is used to synthesize radiolabeled cellodextrins with a degree of polymerization (DP=2-6) from non-radioactive glucose-1-phosphate and radioactive glucose. For cellobiose synthesis, the reaction is carried out at 60°C for 30 min with crude enzyme. After an impluse feed of radiolabeled cellobiose to a Clostridium thermocellum culture, the intracellular sugar levels are measured: the largest amount of radioactivity is cellobiose with lesser amounts of glucose, cellotriose, and cellotetraose, and an average degree of polymerization of intracellular cellodextrins is ca. 2
synthesis
-
the enzyme is useful for production of D-glucose 1-phosphate, an expensive substrate for other enzymatic syntheses
synthesis
the enzyme is useful for synthesis of alkyl beta-glucosides, overview
synthesis
-
enzymatic cellobiose synthesis from starch using two phosphorylases: glucan phosphorylase, from Klebsiella pneumoniae, converts glucose residues in the starch into glucose-1-phosphate, removal of phosphate, and glucose-1-phosphate is incubated with alpha-D-glucose and is converted into cellobiose by recombinant cellobiose phosphorylase, removal of phosphate, 60% cellobiose yield from glucose 1-phosphate and, at least, 23.7% cellobiose yield from starch, method optimization, overview
synthesis
expression of enzyme in Escherichia coli and study on the role of molecular chaperones and growth temperature on the solubilization of enzyme overexpressed in Escherichia coli. The growth of host at low temperature enhances enzyme in soluble fraction. Similarly, induction of target gene at low level of IPTG also yields more enzyme in the soluble fraction. Coexpression of the target gene with molecular chaperones GroESL and KODH does not enhance the solubilization under in vivo conditions
synthesis
-
the enzyme is useful for synthesis of alkyl beta-glucosides, overview
-
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LINKS TO OTHER DATABASES (specific for EC-Number 2.4.1.20)
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