Information on EC 2.7.1.162 - N-acetylhexosamine 1-kinase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
2.7.1.162
-
RECOMMENDED NAME
GeneOntology No.
N-acetylhexosamine 1-kinase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ATP + N-acetyl-D-hexosamine = ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Phosphorylation
SYSTEMATIC NAME
IUBMB Comments
ATP:N-acetyl-D-hexosamine 1-phosphotransferase
This enzyme is involved in the lacto-N-biose I/galacto-N-biose degradation pathway in the probiotic bacterium Bifidobacterium longum. Differs from EC 2.7.1.157, N-acetylgalactosamine kinase, as it can phosphorylate both N-acetylgalactosamine and N-acetylglucosamine at similar rates. Also has some activity with N-acetyl-D-mannosamine, D-talose and D-mannose as substrate. ATP can be replaced by GTP or ITP but with decreased enzyme activity. Requires a divalent cation, with Mg2+ resulting in by far the greatest stimulation of enzyme activity.
CAS REGISTRY NUMBER
COMMENTARY hide
959459-09-7
protein sequence
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
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
ATP + 2-(acetylamino)-2-deoxy-D-allopyranose
ADP + 2-(acetylamino)-2-deoxy-D-allopyranose 1-phosphate
show the reaction diagram
22% phosphorylation yield
-
-
?
ATP + 2-deoxy-2-azido-D-mannose
ADP + 2-deoxy-2-azido-alpha-D-mannose 1-phosphate
show the reaction diagram
ATP + 2-deoxy-2-fluoro-D-mannose
ADP + 2-deoxy-2-fluoro-alpha-D-mannose 1-phosphate
show the reaction diagram
ATP + 2-deoxy-D-glucose
ADP + 2-deoxy-alpha-D-glucose 1-phosphate
show the reaction diagram
ATP + 4- deoxy-N-acetyl-D-glucosamine
ADP + 4-deoxy-N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
-
50% phosphorylation yield
-
-
?
ATP + 4-deoxy-4-azido-N-acetyl-D-galactosamine
ADP + 4-deoxy-4-azido-N-acetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
73% phosphorylation yield
-
-
?
ATP + 4-deoxy-D-mannose
ADP + 4-deoxy-alpha-D-mannose 1-phosphate
show the reaction diagram
ATP + 6-deoxy-6-azido-N-acetyl-D-galactosamine
ADP + 6-deoxy-6-azido-N-acetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
42% phosphorylation yield
-
-
?
ATP + 6-deoxy-6-azido-N-acetyl-D-glucosamine
ADP + 6-deoxy-6-azido-N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ATP + 6-deoxy-6-methyl-N-acetyl-D-glucosamine
ADP + 6-deoxy-6-methyl-N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ATP + 6-deoxy-N-acetyl-D-galactosamine
ADP + 6-deoxy-N-acetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
37% phosphorylation yield
-
-
?
ATP + 6-deoxy-N-acetyl-D-glucosamine
ADP + 6-deoxy-N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
75% phosphorylation yield
-
-
?
ATP + 6-methyl-N-acetyl-D-mannose
ADP + 6-methyl-N-acetyl-alpha-D-mannose 1-phosphate
show the reaction diagram
ATP + D-galactopyranose
ADP + alpha-D-galactopyranose 1-phosphate
show the reaction diagram
-
below 5% phosphorylation yield
-
-
?
ATP + D-galactosamine
ADP + D-galactosamine 1-phosphate
show the reaction diagram
ATP + D-galactose
ADP + D-galactose 1-phosphate
show the reaction diagram
ATP + D-glucosamine
ADP + D-glucosamine 1-phosphate
show the reaction diagram
ATP + D-glucose
ADP + D-glucose 1-phosphate
show the reaction diagram
low activity
-
-
?
ATP + D-mannose
?
show the reaction diagram
ATP + D-mannose
ADP + alpha-D-mannose 1-phosphate
show the reaction diagram
ATP + D-talose
?
show the reaction diagram
phosphorylation at 3% of the activity with N-acetylglucosamine
-
-
?
ATP + mannosamine
ADP + mannosamine 1-phosphate
show the reaction diagram
-
-
-
?
ATP + mannose
ADP + mannose 1-phosphate
show the reaction diagram
-
-
-
?
ATP + N-acetyl-alpha-D-galactosamine
ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-alpha-D-glucosamine
ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-D-galactosamine
ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-D-galactose
ADP + N-acetyl-alpha-D-galactose 1-phosphate
show the reaction diagram
best substrate
-
-
?
ATP + N-acetyl-D-glucosamine
ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-D-glucose
ADP + N-acetyl-alpha-D-glucose 1-phosphate
show the reaction diagram
high activity
-
-
?
ATP + N-acetyl-D-hexosamine
ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-D-mannosamine
?
show the reaction diagram
phosphorylation at 15% of the activity with N-acetylglucosamine
-
-
?
ATP + N-acetyl-D-mannosamine
ADP + ?
show the reaction diagram
-
-
-
?
ATP + N-acetyl-D-mannose
ADP + N-acetyl-alpha-D-mannose 1-phosphate
show the reaction diagram
low activity
-
-
?
ATP + N-azidoacetyl-D-galactosamine
ADP + N-azidoacetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
65% phosphorylation yield
-
-
?
ATP + N-azidoacetyl-D-glucosamine
ADP + N-azidoacetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
87% phosphorylation yield
-
-
?
ATP + N-benzoyl-alpha-D-galactosamine
ADP + N-benzoyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
ATP + N-butanoyl-D-galactosamine
ADP + N-butanoyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
ATP + N-butanoyl-D-glucosamine
ADP + N-butanoyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ATP + N-phenylacetyl-D-galactosamine
ADP + N-phenylacetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
77% phosphorylation yield
-
-
?
ATP + N-phenylacetyl-D-glucosamine
ADP + N-phenylacetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
88% phosphorylation yield
-
-
?
ATP + N-propionyl-D-galactosamine
ADP + N-propionyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
85% phosphorylation yield
-
-
?
ATP + N-propionyl-D-glucosamine
ADP + N-propionyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
86% phosphorylation yield
-
-
?
ATP + N-trifluoroacetyl-alpha-D-galactosamine
ADP + N-trifluoroacetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
-
0.75 microM, 21.8% conversion
-
-
?
ATP + N-trifluoroacetyl-D-glucosamine
ADP + N-trifluoroacetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
-
recombinant enzyme
-
-
?
GTP + N-acetyl-alpha-D-glucosamine
GDP + N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
GTP shows about 45% of the activity with ATP
-
-
?
ITP + N-acetyl-alpha-D-glucosamine
IDP + N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ITP shows about 30% of the activity with ATP
-
-
?
additional information
?
-
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
ATP + N-acetyl-alpha-D-galactosamine
ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-alpha-D-glucosamine
ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
show the reaction diagram
ATP + N-acetyl-D-hexosamine
ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
1 mM, 1% relative activity compared to Mg2+
Co2+
1 mM, 21% relative activity compared to Mg2+; 21% of the activity with Mg2+
MgCl2
-
10 mM are included in the assay medium
Mn2+
1 mM, 7% relative activity compared to Mg2+
Ni2+
1 mM, 4% relative activity compared to Mg2+
Zn2+
1 mM, 8% relative activity compared to Mg2+
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
isopropyl-1-thio-beta-D-galactoside
induction of protein expression at a final concentration of 0.5 mM
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.005 - 0.9
ATP
0.065 - 0.09
N-acetyl-alpha-D-galactosamine
0.06 - 0.118
N-acetyl-alpha-D-glucosamine
0.35 - 3.14
N-acetyl-D-glucosamine
additional information
additional information
-
summary of ITC analysis of wild-type NahK and mutants
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12 - 16.3
ATP
0.46 - 6.08
N-acetyl-alpha-D-galactosamine
0.95 - 1.21
N-acetyl-alpha-D-glucosamine
0.11 - 13.6
N-acetyl-D-glucosamine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.8 - 11.8
ATP
4
5.1 - 7.1
N-acetyl-alpha-D-galactosamine
2057
15.8 - 18.3
N-acetyl-alpha-D-glucosamine
1537
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.001
D-glucose, substrate donor specificity
0.002
D-mannosamine, substrate donor specificity
0.004
D-glucosamine, substrate donor specificity
0.007
2-deoxyglucose, substrate donor specificity
0.024
D-mannose, substrate donor specificity
0.049
D-talose, substrate donor specificity
0.22
N-acetyl-D-mannosamine, substrate donor specificity
0.48
ITP, substrate donor specificity
0.65
GTP, substrate donor specificity
0.89
N-acetyl-D-galactosamine, substrate donor specificity
1.48
ATP, substrate donor specificity
1.49
N-acetyl-D-glucosamine, substrate donor specificity
additional information
Incubation of 4 microM LnpB protein with 10 mM N-acetyl-alpha-D-glucosamine 6-sulfate in 0.1 M MOPS buffer (pH 7.5) at 30°C for 30 min shows that the protein have no hydrolytic activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bifidobacterium longum subsp. infantis (strain ATCC 15697 / DSM 20088 / JCM 1222 / NCTC 11817 / S12)
Bifidobacterium longum subsp. infantis (strain ATCC 15697 / DSM 20088 / JCM 1222 / NCTC 11817 / S12)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
Bifidobacterium longum subsp. longum (strain ATCC 15707 / DSM 20219 / JCM 1217 / NCTC 11818 / E194b)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant His-tagged enzyme in complex with substrates GalNAc, GlcNAc-1P, GlcNAc/AMPPNP (a nonhydrolyzable ATP analogue) and GlcNAc-1P/ADP, hanging drop vapour diffusion method, pre-incubation of SeMet-substituted/wild-type NahK with GlcNAc (20fold excess), GalNAc (20fold excess) or GlcNAc-1P (10fold excess), and mixing of 20 mg/ml protein in 50 mM HEPES, pH 7.5, with a crystallization solution containing 20% w/v PEG 3350, 0.1 M Bis-Tris, pH 5.5, crystals of NahK-GlcNAc-AMPPNP are obtained by soaking NahK-GlcNAc crystals with 10 mM AMPPNP and 20 mM MgCl2 for 15 min, or the crystals of NahK-GlcNAc-1P-ADP by soaking NahK-GlcNAc-1P crystals with 5 mM ADP and 10 mM MgCl2 for 30 min, crystallization time is 10-14 days, 20°C, X-ray diffraction structure determination and analysis at 1.72-2.15 A resolution, molecular replacement method, modelling
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purified recombinant wild-type enzyme in apoform, or SeMet-labeled NahK complexed with ATP, sitting drop vapor diffusion method, mixing of 500 nl protein solution containing 25 mg/mL NahK and with or without 10 mM ATP with an equal volume of reservoir solution containing 0.2 M magnesium acetate, 0.1 M sodium cacodylate, pH 6.5, and 20% PEG 8000, for ADP complex crystals, 10 mM ADP is used instead of ATP, 20°C, X-ray diffraction structure determination and analysis at 1.80-2.05 A resolution
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purified recombinant His-tagged enzyme in complex with substrates GlcNAc, and GlcNAc/AMPPNP (a nonhydrolyzable ATP analogue), hanging drop vapour diffusion method, mixing of 20 mg/ml protein in 50 mM HEPES, pH 7.5, with crystallization solution containing 1.5 M Li2SO4, 0.1 M sodium acetate, pH 5.5, 2 days, crystals of the NahKATCC15697-GlcNAc-AMPPNP complex are obtained by soaking NahK-GlcNAc complex crystals with 5 mM AMPPNP and 10 mM MgCl2 for 1.5 h, 20°C, X-ray diffraction structure determination and analysis at 1.47-1.90 A resolution, molecular replacement method, modelling
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 9.5
stability at 30°C; stable
677700
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50
pH 8.5, 30 min, 50% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
loses half of enzyme activity during incubation for 30 min at 50°C in 0.1 M Tris buffer (pH 8.5)
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; The transformant is cultivated and protein expression is induced by the addition of isopropyl-1-thio-beta-D-galactoside. The cells are harvested by centrifugation and sonicated. Cell debris is removed by centrifugation and enzyme is purified on an Ni-nitrilotriacetic acid agarose gel.
recombinant C-terminally His6-tagged wild-type and SeMet-labeled NahK residues 1-359 enzymes from Escherichia coli by nickel affinity chromatography and gel filtration
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recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Escherichia coli strains select96 and BL21(DE3) are used as hosts for cloning and expression.; expression in Escherichia coli
expression in Escherichia coli
gene lnpB, recombinant expression of C-terminally His6-tagged NahK residues 1-359 in Escherichia coli strain BL21 CodonPlus (DE3)-RIL, expression of the (SeMet)-labeled protein in Escherichia coli strain B834 (DE3)
-
gene lnpB, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
recombinant expression in Escherichia coli strain BL21(DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D208 A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
D208A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
D208A/K210A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
D208A/K210L
-
site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type
D208L
-
site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type
D228A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
I146E
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
I146E/T231E
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
K210E
-
site-directed mutagenesis, the mutant shows reduced highly activity and altered substrate specificity compared to the wild-type
K210L
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
N213A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
Q48A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
R306A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
R306A/Y317A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
R306E
-
site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type
R306E/Y317F
-
site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type
T231 E
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
Y317A
-
site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type
Y317F
-
site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type
D208A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
-
D228A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
-
K210A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
-
N213A
-
site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme
-
Q48A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
-
R306A
-
site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type
-
R306E
-
site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type
-
Y317A
-
site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type
-
Y317F
-
site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type
-
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis