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Literature summary extracted from

  • Wang, K.C.; Lyu, S.Y.; Liu, Y.C.; Chang, C.Y.; Wu, C.J.; Li, T.L.
    Insights into the binding specificity and catalytic mechanism of N-acetylhexosamine 1-phosphate kinases through multiple reaction complexes (2014), Acta Crystallogr. Sect. D, 70, 1401-1410.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.1.162 gene lnpB, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Bifidobacterium longum subsp. infantis
2.7.1.162 gene lnpB, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) Bifidobacterium longum

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.1.162 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 Bifidobacterium longum
2.7.1.162 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 Bifidobacterium longum subsp. infantis

Protein Variants

EC Number Protein Variants Comment Organism
2.7.1.162 D208 A site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 D208A/K210A site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 D208A/K210L site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 D208L site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 I146E site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 I146E/T231E site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 K210A site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 K210E site-directed mutagenesis, the mutant shows reduced highly activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 K210L site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 Q48A site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 R306A site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 R306A/Y317A site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 R306E site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 R306E/Y317F site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 T231 E site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 Y317A site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type Bifidobacterium longum
2.7.1.162 Y317F site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type Bifidobacterium longum

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.7.1.162 additional information
-
additional information summary of ITC analysis of wild-type NahK and mutants Bifidobacterium longum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.1.162 Mg2+ required, binding structure, overview Bifidobacterium longum subsp. infantis
2.7.1.162 Mg2+ required, binding structure, overview Bifidobacterium longum

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.1.162 42500
-
gel-filtration and analytical ultracentrifugation Bifidobacterium longum subsp. infantis
2.7.1.162 42500
-
gel-filtration and analytical ultracentrifugation 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 subsp. infantis
-
ADP + N-acetyl-alpha-D-hexosamine 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 subsp. infantis ATCC 15697
-
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
-
2.7.1.162 Bifidobacterium longum subsp. infantis
-
gene lnpB
-
2.7.1.162 Bifidobacterium longum subsp. infantis ATCC 15697
-
gene lnpB
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.1.162 recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration Bifidobacterium longum subsp. infantis
2.7.1.162 recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) 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 regio- and stereoselectivity, catalytic mechanism, structure-function analysis, overview. Nucleophilic attack on the gamma-phosphate atom in a bond-breaking/bond formation manner of transfer (an SN2-like reaction). The carboxyl group of residue Asp208 within hydrogen-bond distance of C1 OH of GlcNAc/GalNAc is likely to serve as the active-site general base Bifidobacterium longum subsp. infantis
2.7.1.162 ATP + N-acetyl-D-hexosamine = ADP + N-acetyl-alpha-D-hexosamine 1-phosphate regio- and stereoselectivity, catalytic mechanism, structure-function analysis, overview. Nucleophilic attack on the gamma-phosphate atom in a bond-breaking/bond formation manner of transfer (an SN2-like reaction). The carboxyl group of residue Asp208 within hydrogen-bond distance of C1 OH of GlcNAc/GalNAc is likely to serve as the active-site general base Bifidobacterium longum

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.1.162 ATP + D-galactosamine low activity Bifidobacterium longum ADP + D-galactosamine 1-phosphate
-
?
2.7.1.162 ATP + D-galactosamine low activity Bifidobacterium longum JCM 1217 ADP + D-galactosamine 1-phosphate
-
?
2.7.1.162 ATP + D-galactose low activity Bifidobacterium longum ADP + D-galactose 1-phosphate
-
?
2.7.1.162 ATP + D-galactose low activity Bifidobacterium longum JCM 1217 ADP + D-galactose 1-phosphate
-
?
2.7.1.162 ATP + D-glucosamine
-
Bifidobacterium longum ADP + D-glucosamine 1-phosphate
-
?
2.7.1.162 ATP + D-glucose low activity Bifidobacterium longum ADP + D-glucose 1-phosphate
-
?
2.7.1.162 ATP + D-mannosamine
-
Bifidobacterium longum ADP + alpha-D-mannosamine 1-phosphate
-
?
2.7.1.162 ATP + mannose
-
Bifidobacterium longum ADP + mannose 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-galactosamine
-
Bifidobacterium longum subsp. infantis ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-galactosamine best substrate Bifidobacterium longum ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-galactosamine best substrate Bifidobacterium longum JCM 1217 ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-galactosamine
-
Bifidobacterium longum subsp. infantis ATCC 15697 ADP + N-acetyl-alpha-D-galactosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-glucosamine
-
Bifidobacterium longum subsp. infantis ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-glucosamine high activity Bifidobacterium longum ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-glucosamine high activity Bifidobacterium longum JCM 1217 ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-glucosamine
-
Bifidobacterium longum subsp. infantis ATCC 15697 ADP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-hexosamine
-
Bifidobacterium longum subsp. infantis ADP + N-acetyl-alpha-D-hexosamine 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 subsp. infantis ATCC 15697 ADP + N-acetyl-alpha-D-hexosamine 1-phosphate
-
?
2.7.1.162 ATP + N-acetyl-D-mannosamine low activity Bifidobacterium longum ADP + N-acetyl-alpha-D-mannosamine 1-phosphate
-
?
2.7.1.162 additional information substrate binding structures, overview Bifidobacterium longum subsp. infantis ?
-
?
2.7.1.162 additional information substrate binding structures, overview. No activity with glucuronic acid, galacturonic acid, 6-azidoglucose, 6-GlcNAc, 3-glucosamine, 4-glucosamine, and 6-glucosamine Bifidobacterium longum ?
-
?
2.7.1.162 additional information substrate binding structures, overview. No activity with glucuronic acid, galacturonic acid, 6-azidoglucose, 6-GlcNAc, 3-glucosamine, 4-glucosamine, and 6-glucosamine Bifidobacterium longum JCM 1217 ?
-
?
2.7.1.162 additional information substrate binding structures, overview Bifidobacterium longum subsp. infantis ATCC 15697 ?
-
?

Subunits

EC Number Subunits Comment Organism
2.7.1.162 monomer 1 * 42500, SDS-PAGE Bifidobacterium longum
2.7.1.162 monomer 1 * 42500, SDS-PAGE, enzyme NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The N- and C-terminal domains of NahK comprise residues 1-103 and 117-359, respectively. The N-terminal domain contains a characteristic five-stranded antiparallel beta-sheet flanked by two alpha-helices Bifidobacterium longum subsp. infantis
2.7.1.162 More enzyme NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The N- and C-terminal domains of NahK comprise residues 1-103 and 117-359, respectively. The N-terminal domain contains a characteristic five-stranded antiparallel beta-sheet flanked by two alpha-helices Bifidobacterium longum

Synonyms

EC Number Synonyms Comment Organism
2.7.1.162 hexosamine kinase
-
Bifidobacterium longum subsp. infantis
2.7.1.162 hexosamine kinase
-
Bifidobacterium longum
2.7.1.162 N-acetylhexosamine 1-phosphate kinase
-
Bifidobacterium longum subsp. infantis
2.7.1.162 N-acetylhexosamine 1-phosphate kinase
-
Bifidobacterium longum
2.7.1.162 NahK
-
Bifidobacterium longum subsp. infantis
2.7.1.162 NahK
-
Bifidobacterium longum
2.7.1.162 NahKATCC15697
-
Bifidobacterium longum subsp. infantis

Temperature Optimum [°C]

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

pH Optimum

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

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.1.162 ATP ATP/ADP binding structures, overview Bifidobacterium longum subsp. infantis
2.7.1.162 ATP ATP/ADP binding structures, overview Bifidobacterium longum

General Information

EC Number General Information Comment Organism
2.7.1.162 evolution the enzyme belongs to the sugar kinase-Hsp70-actin superfamily Bifidobacterium longum subsp. infantis
2.7.1.162 evolution the enzyme belongs to the sugar kinase-Hsp70-actin superfamily Bifidobacterium longum
2.7.1.162 metabolism the enzyme is part of the LNB/GNB pathway in bifidobacteria, overview Bifidobacterium longum subsp. infantis
2.7.1.162 metabolism the enzyme is part of the LNB/GNB pathway in bifidobacteria, overview Bifidobacterium longum