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Information on EC 2.7.1.232 - levoglucosan kinase
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EC Tree
2.7.1.232 levoglucosan kinaseIUBMB Comments
Levoglucosan is formed from the pyrolysis of carbohydrates such as starch and cellulose and is an important molecular marker for pollution from biomass burning. The enzyme, found in yeast and fungi, requires a magnesium ion. cf. EC 1.1.1.425, levoglucosan dehydrogenase.
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Word Map
- 2.7.1.232
-
pyrolysis
-
starkeyi
-
lipomyces
-
anhydrosugars
-
cellulosic
-
bio-oils
-
niger
-
6-phosphate
-
lignocellulosic
- glucose-6-phosphate
-
bioconversion
-
1,6-anhydro-n-acetylmuramic
- magnesium
-
gibberella
-
pyrolysate
-
in-gel
- adp
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biofuel
The expected taxonomic range for this enzyme is: Eukaryota, Bacteria
Reaction Schemes
Synonyms
levoglucosan kinase, 
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
levoglucosan kinase
LGK
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
ATP + levoglucosan + H2O = ADP + D-glucose 6-phosphate
-
-
-
-
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
catalytic mechanism, molecular docking, dynamics simulation
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ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
catalytic mechanism, molecular docking, dynamics simulation
-
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
mechanism of 1,6-anhydro bond cleavage, the enzyme binds levoglucosan in two distinct orientations, in addition to cleavage of the 1,6-anhydro ring, the pyranose ring of the levoglucosan is opened when the anomeric carbon of levoglucosan migrates from its position in a 1C4 to a 4C1 conformation, increased conformational strain during this reaction results in the linear form of G6P as the initial product, which then equilibrates to a mixture of both anomeric forms, structure-function analysis, detailed overview
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
the catalytic mechanism involves both cleavage of the 1,6-intramolecular linkage as well as phosphorylation
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ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
the catalytic mechanism involves both cleavage of the 1,6-intramolecular linkage as well as phosphorylation
-
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
mechanism of 1,6-anhydro bond cleavage, the enzyme binds levoglucosan in two distinct orientations, in addition to cleavage of the 1,6-anhydro ring, the pyranose ring of the levoglucosan is opened when the anomeric carbon of levoglucosan migrates from its position in a 1C4 to a 4C1 conformation, increased conformational strain during this reaction results in the linear form of G6P as the initial product, which then equilibrates to a mixture of both anomeric forms, structure-function analysis, detailed overview
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-
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O = ADP + 6-phospho-alpha-D-glucopyranose
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-
-
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SYSTEMATIC NAME
IUBMB Comments
ATP:1,6-anhydro-beta-D-glucopyranose 6-phosphotransferase (hydrolyzing)
Levoglucosan is formed from the pyrolysis of carbohydrates such as starch and cellulose and is an important molecular marker for pollution from biomass burning. The enzyme, found in yeast and fungi, requires a magnesium ion. cf. EC 1.1.1.425, levoglucosan dehydrogenase.
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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
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + 6-phospho-alpha-D-glucopyranose
-
-
-
-
?
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + 6-phospho-alpha-D-glucopyranose
-
-
-
-
?
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + alpha-D-glucose-6-phosphate
-
-
-
ir
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + alpha-D-glucose-6-phosphate
1,6-anhydro-beta-D-glucopyranose is levoglucosan. The enzyme is highly specific for levoglucosan
-
-
ir
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
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-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
FAB-mass spectrometric method for product identification
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl2
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl2
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
FAB-mass spectrometric method for product identification
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
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-
reaction products ADP and glucose 6-phosphate are measured by a pyruvatekinase/lactate dehydrogenase coupling system as well as through the use of 14C labeled levoglucosan and thin layer chromatography techniques
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?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
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-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
in addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom
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-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
structure-function analysis and reaction mechanism
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-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
structure-function analysis and reaction mechanism
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
in addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom
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-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
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-
reaction products ADP and glucose 6-phosphate are measured by a pyruvatekinase/lactate dehydrogenase coupling system as well as through the use of 14C labeled levoglucosan and thin layer chromatography techniques
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?
additional information
?
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-
substrate specificity for levoglucosan, not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame is specific for recognition by levoglucosan kinase
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-
?
additional information
?
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-
substrate specificity for levoglucosan, not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame is specific for recognition by levoglucosan kinase
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-
?
additional information
?
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although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK, EC 2.7.1.170), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. A second alternate binding orientation for levoglucosan is found, and in these structures, ADP binds with lower affinity, explaining the high Km of enzyme LGK for levoglucosan. LGK binds the reaction product ADP through multiple hydrogen bonds, including bonds between the adenyl moiety and Asp237, between the nucleotide ribose and Asp221, and between the ADP phosphates and protein residues Ser24, Gly189, and Gly328, mechanism of 1,6-anhydro bond cleavage, overview
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-
?
additional information
?
-
no enzymatic back reaction from alpha-D-glucose-6-phosphate and ADP is observed. The enzyme also shows no activity with ADP plus 6-phospho-alpha-D-glucopyranosyl fluoride or 6-phospho-D-glucal
-
-
-
additional information
?
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no enzymatic back reaction from alpha-D-glucose-6-phosphate and ADP is observed. The enzyme also shows no activity with ADP plus 6-phospho-alpha-D-glucopyranosyl fluoride or 6-phospho-D-glucal
-
-
-
additional information
?
-
although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK, EC 2.7.1.170), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. A second alternate binding orientation for levoglucosan is found, and in these structures, ADP binds with lower affinity, explaining the high Km of enzyme LGK for levoglucosan. LGK binds the reaction product ADP through multiple hydrogen bonds, including bonds between the adenyl moiety and Asp237, between the nucleotide ribose and Asp221, and between the ADP phosphates and protein residues Ser24, Gly189, and Gly328, mechanism of 1,6-anhydro bond cleavage, overview
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-
?
additional information
?
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enzme LGK shows specificity for the levoglucosan sugar, showing activity for only this anhydrosugar and no activity for galactosan or maltosan and only very weak activity for mannosan (1% relative activity) that also contain the same 1,6-anhydro intramolecular linkage as levoglucosan, demonstrating that the nature of the pyranose frame is also important for substrate recognition by enzyme LGK
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-
?
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NATURAL SUBSTRATE
NATURAL 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
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + alpha-D-glucose-6-phosphate
-
-
-
ir
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + 6-phospho-alpha-D-glucopyranose
-
-
-
-
?
ATP + 1,6-anhydro-beta-D-glucopyranose + H2O
ADP + 6-phospho-alpha-D-glucopyranose
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl2
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
direct formation of glucose 6-phosphate from levoglucosan in the presence of ATP and MgCl2
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
?
ATP + levoglucosan + H2O
ADP + D-glucopyranose 6-phosphate
-
-
-
-
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mg2+
Mn2+
the enzyme requires Mg2+ or Mn2+, apparent binding of two magnesium ions in the active site showing ideal octahedral binding of the metals. The first of the bound metals, designated M1, forms an electrostatic interaction with the beta-phosphate, and its positioning suggests that it plays a direct role in phosphoryl transfer. The second of these metals, designated M2, likely plays a key role in coordinating the position of the alpha- and beta-phosphates since it binds to both of these phosphates, although a role in modulation of electrostatic charges is also plausible
Mg2+
required, the enzyme binds two magnesium ions in the active site, four manganese atoms in the dimeric structure, that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. The magnesium ions are observed in ideal octahedral coordination with Glu362 and Asp26, several water molecules, and the ADP, overview. The first of the bound metals, designated M1, forms an electrostatic interaction with the beta-phosphate, and its positioning suggests that it plays a direct role in phosphoryl transfer. The second of these metals, designated M2, likely plays a role in coordinating the position of the alpha- and beta-phosphates because it binds to both of these phosphates, whereas a role in modulation of electrostatic charges is also plausible
Mg2+
the enzyme requires Mg2+ or Mn2+, apparent binding of two magnesium ions in the active site showing ideal octahedral binding of the metals. The first of the bound metals, designated M1, forms an electrostatic interaction with the beta-phosphate, and its positioning suggests that it plays a direct role in phosphoryl transfer. The second of these metals, designated M2, likely plays a key role in coordinating the position of the alpha- and beta-phosphates since it binds to both of these phosphates, although a role in modulation of electrostatic charges is also plausible
Mg2+
the enzyme contains two Mg2+ ions. Using 4 and 8 mM Mg2+, the activity is highest at a Mg2+/ATP ratio of 2 and drastically drops if the ratio is less than 1. Employing a constant ATP concentration (2.5 mM), the enzyme activity increases with the Mg2+ concentration until a Mg2+/ATP ratio of 8 is reached at 20 mM MgCl2
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
48
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
56
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
70
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
80
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
84
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
85
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
95
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
102
1,6-anhydro-beta-D-glucopyranose
-
pH and temperature not specified in the publication
119
1,6-anhydro-beta-D-glucopyranose
pH and temperature not specified in the publication
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Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
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IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.05
gentiobiose
Sporidiobolus salmonicolor
-
pH and temperature not specified in the publication
0.015
Mg-ADP
Sporidiobolus salmonicolor
-
pH and temperature not specified in the publication
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
30
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
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SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
additional information
-
inductive synthesis of levoglucosan kinase, the organism grows well on levoglucosan as sole carbon source, fermenting it into citric acid with a yield comparable to that on glucose
brenda
additional information
-
inductive synthesis of levoglucosan kinase, the organism grows well on levoglucosan as sole carbon source, fermenting it into citric acid with a yield comparable to that on glucose
-
brenda
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GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
additional information
evolution
-
phylogenetic analysis and comparison of 1,6-anhydro-N-acetylmuramic acid kinase with levoglucan kinase and AnmK-like enzymes, molecular docking, dynamics simulation, and homology modelling, overview. AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively
evolution
-
phylogenetic analysis and comparison of levoglucan kinase with 1,6-anhydro-N-acetylmuramic acid kinase and AnmK-like enzymes, molecular docking, dynamics simulation, and homology modelling, overview. AnmK and LGK are conserved proteins, and 187Asp, 212Asp are enzymatic residues, respectively
additional information
three dimensional structure analysis, comparison of structure and mechanism with 1,6-anhydro-N-acetylmuramic acid kinase and AnmK-like enzymes, overview
additional information
-
three dimensional structure analysis, comparison with 1,6-anhydro-N-acetylmuramic acid kinase and AnmK-like enzymes, overview
additional information
-
three dimensional structure analysis, comparison of structure and mechanism with 1,6-anhydro-N-acetylmuramic acid kinase and AnmK-like enzymes, overview
-
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). LGK_LIPST
439
0
48372
Swiss-Prot
other Location (Reliability: 3)
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MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme, hanging drop vapor diffusion method, dimeric enzyme: mixing of equal volumes of 7 mg/ml protein in 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5mM TCEP, and 20mM Tris, pH 7.5, with reservoir solution containing 18% PEG 4000, 100 mM sodium acetate, 100 mM Tris, pH 7.2, for the free enzyme and with reservoir solution containing 50 mM NaCl, 2 mM ADP, 4 mM MgCl2, 0.5 mM TCEP, 1 mM aluminum nitrate, 10 mM sodium fluoride, 20 mM Tris, pH 7.5, for the complexed enzyme. Monomeric enzyme: 9 mg/ml protein in 50 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, and 200 mM levoglucosan is mixed with reservoir buffer containing 1.8 M ammonium sulfate, HEPES, pH 7.0, and 100 mM sodium acetate for th free enzyme, and 25 mg/ml protein in 100 mM NaCl, 0.5 mM TCEP, 20 mM Tris, pH 7.5, 2 mM ADP, 4 mM MgCl2, 1 mM aluminum nitrate, and 10 mM sodium fluoride, with reservoir buffer containing 1.3 M sodium malonate, 93 mM Bis-Tris propane, pH 7.0, for the complexed enzyme, soaking of resultant crystals in the drop with 200 mM levoglucosan. X-ray diffraction structure determination and analysis at 1.5-2.0 A resolution
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
LGK derived from Lipomyces starkeyi is an interesting target for efforts in advanced protein engineering, single-site saturation mutagenesis coupled with selection of the LGK variants using deep sequencing approaches is used to develop LGK constructs with enhanced thermal stability and catalytic efficiency
additional information
-
LGK derived from Lipomyces starkeyi is an interesting target for efforts in advanced protein engineering, single-site saturation mutagenesis coupled with selection of the LGK variants using deep sequencing approaches is used to develop LGK constructs with enhanced thermal stability and catalytic efficiency
-
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
6 - 10
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25 - 37
at 30°C and pH 7.5, all enzyme activity is lost within 2 h. A temperature decrease to 25°C and the addition of 1 mg/ml bovine serum albumin enhance the enzyme's half-life to approximately 4 h. The enzyme's melting temperature is 37°C
additional information
-
levoglucosan can protect the enzyme from thermal inactivation
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
native enzyme by ion exchange chromatography
native enzyme partially by ammonium sulfate fractionation and anion exchange chromatography
-
native enzyme to apparent homogeneity by using a combination of seven purification steps
-
native enzymepartially by ammonium sulfate fractionation and ion exchange chromatography
-
recombinant enzyme from Escherichia coli strain BL21(DE3) GOLD by metal affinity chromatography, gel filtration, and ultrafiltration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE