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Literature summary extracted from

  • Sato, M.; Arakawa, T.; Nam, Y.W.; Nishimoto, M.; Kitaoka, M.; Fushinobu, S.
    Open-close structural change upon ligand binding and two magnesium ions required for the catalysis of N-acetylhexosamine 1-kinase (2015), Biochim. Biophys. Acta, 1854, 333-340.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.162 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) Bifidobacterium longum

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.162 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 Bifidobacterium longum

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.162 D208A site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme Bifidobacterium longum
2.7.1.162 D228A site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme Bifidobacterium longum
2.7.1.162 K210A site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme Bifidobacterium longum
2.7.1.162 N213A site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme Bifidobacterium longum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.1.162 0.005
-
 ATP pH 8.0, 37°C, recombinant mutant K210A, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 0.038
-
 ATP pH 8.0, 37°C, recombinant mutant N213A at 50 mM MgCl2 Bifidobacterium longum
2.7.1.162 0.1
-
 ATP pH 8.0, 37°C, recombinant wild-type enzyme, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 0.35
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant mutant K210A, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 0.9
-
 ATP pH 8.0, 37°C, recombinant mutant N213A at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 1.8
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant wild-type enzyme, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 2.5
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant mutant N213A at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 3.14
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant mutant N213A at 50 mM MgCl2 Bifidobacterium longum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.162 Mg2+ requirement of the second magnesium ion, the enzyme has two magnesium binding sites, Mg1 and Mg2, structure overview Bifidobacterium longum

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.1.162 ATP + N-acetyl-D-hexosamine Bifidobacterium longum
-
 ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
-
 ?
2.7.1.162 ATP + N-acetyl-D-hexosamine Bifidobacterium longum JCM 1217
-
 ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.1.162 Bifidobacterium longum E8MF12 gene lnpB
-
2.7.1.162 Bifidobacterium longum JCM 1217 E8MF12 gene lnpB
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.162 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 Bifidobacterium longum

Reaction

EC Number Reaction Comment Organism Reaction ID
2.7.1.162 ATP + N-acetyl-D-hexosamine = ADP + N-acetyl-alpha-D-hexosamine 1-phosphate open–close conformational change at the active site, structure overview Bifidobacterium longum
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.162 ATP + N-acetyl-D-glucosamine
-
 Bifidobacterium longum ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 ?
2.7.1.162 ATP + N-acetyl-D-glucosamine
-
 Bifidobacterium longum JCM 1217 ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 ?
2.7.1.162 ATP + N-acetyl-D-hexosamine
-
 Bifidobacterium longum ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
-
 ?
2.7.1.162 ATP + N-acetyl-D-hexosamine
-
 Bifidobacterium longum JCM 1217 ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.1.162 dimer
-
 Bifidobacterium longum
2.7.1.162 More enzyme domain architecture, overview Bifidobacterium longum

Synonyms

EC Number Synonyms Comment Organism
2.7.1.162 hexosamine kinase
-
 Bifidobacterium longum
2.7.1.162 N-acetylhexosamine 1-phosphate kinase
-
 Bifidobacterium longum
2.7.1.162 NahK
-
 Bifidobacterium longum

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.1.162 37
-
 assay at Bifidobacterium longum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.7.1.162 0.11
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant mutant K210A, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 0.12
-
 ATP pH 8.0, 37°C, recombinant mutant K210A, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 5.9
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant mutant N213A at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 6.4
-
 ATP pH 8.0, 37°C, recombinant mutant N213A at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 8.8
-
 ATP pH 8.0, 37°C, recombinant mutant N213A at 50 mM MgCl2 Bifidobacterium longum
2.7.1.162 9.9
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant wild-type enzyme, at 5.0 mM MgCl2 Bifidobacterium longum
2.7.1.162 13.6
-
 N-acetyl-D-glucosamine pH 8.0, 37°C, recombinant mutant N213A at 50 mM MgCl2 Bifidobacterium longum
2.7.1.162 16.3
-
 ATP pH 8.0, 37°C, recombinant wild-type enzyme, at 5.0 mM MgCl2 Bifidobacterium longum

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.1.162 8
-
 assay at Bifidobacterium longum

General Information

EC Number General Information Comment Organism
2.7.1.162 additional information nucleotide binding site structures of NahK-ATP and Nahk-ADP, overview Bifidobacterium longum