Literature summary for 2.7.1.162 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.

Search for more literature for 2.7.1.162

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

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

KM Value [mM]

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

Metals/Ions

Metals/Ions	Comment	Organism	Structure
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)

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

Organism

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

Purification (Commentary)

Purification (Comment)	Organism
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

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

Substrates and Products (Substrate)

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

Subunits

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

Synonyms

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

Temperature Optimum [°C]

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

Turnover Number [1/s]

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

pH Optimum

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

General Information

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