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 (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 | 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 Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 | 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Bifidobacterium longum | E8MF12 | gene lnpB | - |
Bifidobacterium longum JCM 1217 | E8MF12 | gene lnpB | - |
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 | 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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
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 | Comment | Organism |
---|---|---|
dimer | - |
Bifidobacterium longum |
More | enzyme domain architecture, overview | Bifidobacterium longum |
Synonyms | Comment | Organism |
---|---|---|
hexosamine kinase | - |
Bifidobacterium longum |
N-acetylhexosamine 1-phosphate kinase | - |
Bifidobacterium longum |
NahK | - |
Bifidobacterium longum |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Bifidobacterium longum |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Bifidobacterium longum |
General Information | Comment | Organism |
---|---|---|
additional information | nucleotide binding site structures of NahK-ATP and Nahk-ADP, overview | Bifidobacterium longum |