Cloned (Comment) | Organism |
---|---|
gene lnpB, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) | Bifidobacterium longum subsp. infantis |
gene lnpB, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3) | Bifidobacterium longum |
Crystallization (Comment) | Organism |
---|---|
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 |
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 | Comment | Organism |
---|---|---|
D208 A | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
D208A/K210A | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
D208A/K210L | site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
D208L | site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
I146E | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
I146E/T231E | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
K210A | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
K210E | site-directed mutagenesis, the mutant shows reduced highly activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
K210L | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
Q48A | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
R306A | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
R306A/Y317A | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
R306E | site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type | Bifidobacterium longum |
R306E/Y317F | site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type | Bifidobacterium longum |
T231 E | site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type | Bifidobacterium longum |
Y317A | site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type | Bifidobacterium longum |
Y317F | site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type | Bifidobacterium longum |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | summary of ITC analysis of wild-type NahK and mutants | Bifidobacterium longum |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required, binding structure, overview | Bifidobacterium longum subsp. infantis | |
Mg2+ | required, binding structure, overview | Bifidobacterium longum |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
42500 | - |
gel-filtration and analytical ultracentrifugation | Bifidobacterium longum subsp. infantis |
42500 | - |
gel-filtration and analytical ultracentrifugation | Bifidobacterium longum |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + N-acetyl-D-hexosamine | Bifidobacterium longum subsp. infantis | - |
ADP + N-acetyl-alpha-D-hexosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-hexosamine | Bifidobacterium longum | - |
ADP + N-acetyl-alpha-D-hexosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-hexosamine | Bifidobacterium longum subsp. infantis ATCC 15697 | - |
ADP + N-acetyl-alpha-D-hexosamine 1-phosphate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bifidobacterium longum | E8MF12 | gene lnpB | - |
Bifidobacterium longum JCM 1217 | E8MF12 | gene lnpB | - |
Bifidobacterium longum subsp. infantis | - |
gene lnpB | - |
Bifidobacterium longum subsp. infantis ATCC 15697 | - |
gene lnpB | - |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Bifidobacterium longum subsp. infantis |
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) 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 | 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 | |
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 | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
ATP + D-galactosamine | low activity | Bifidobacterium longum | ADP + D-galactosamine 1-phosphate | - |
? | |
ATP + D-galactosamine | low activity | Bifidobacterium longum JCM 1217 | ADP + D-galactosamine 1-phosphate | - |
? | |
ATP + D-galactose | low activity | Bifidobacterium longum | ADP + D-galactose 1-phosphate | - |
? | |
ATP + D-galactose | low activity | Bifidobacterium longum JCM 1217 | ADP + D-galactose 1-phosphate | - |
? | |
ATP + D-glucosamine | - |
Bifidobacterium longum | ADP + D-glucosamine 1-phosphate | - |
? | |
ATP + D-glucose | low activity | Bifidobacterium longum | ADP + D-glucose 1-phosphate | - |
? | |
ATP + D-mannosamine | - |
Bifidobacterium longum | ADP + alpha-D-mannosamine 1-phosphate | - |
? | |
ATP + mannose | - |
Bifidobacterium longum | ADP + mannose 1-phosphate | - |
? | |
ATP + N-acetyl-D-galactosamine | - |
Bifidobacterium longum subsp. infantis | ADP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-galactosamine | best substrate | Bifidobacterium longum | ADP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-galactosamine | best substrate | Bifidobacterium longum JCM 1217 | ADP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-galactosamine | - |
Bifidobacterium longum subsp. infantis ATCC 15697 | ADP + N-acetyl-alpha-D-galactosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-glucosamine | - |
Bifidobacterium longum subsp. infantis | ADP + N-acetyl-alpha-D-glucosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-glucosamine | high activity | Bifidobacterium longum | ADP + N-acetyl-alpha-D-glucosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-glucosamine | high activity | Bifidobacterium longum JCM 1217 | ADP + N-acetyl-alpha-D-glucosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-glucosamine | - |
Bifidobacterium longum subsp. infantis ATCC 15697 | ADP + N-acetyl-alpha-D-glucosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-hexosamine | - |
Bifidobacterium longum subsp. infantis | ADP + N-acetyl-alpha-D-hexosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-hexosamine | - |
Bifidobacterium longum | ADP + N-acetyl-alpha-D-hexosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-hexosamine | - |
Bifidobacterium longum subsp. infantis ATCC 15697 | ADP + N-acetyl-alpha-D-hexosamine 1-phosphate | - |
? | |
ATP + N-acetyl-D-mannosamine | low activity | Bifidobacterium longum | ADP + N-acetyl-alpha-D-mannosamine 1-phosphate | - |
? | |
additional information | substrate binding structures, overview | Bifidobacterium longum subsp. infantis | ? | - |
? | |
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 | ? | - |
? | |
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 | ? | - |
? | |
additional information | substrate binding structures, overview | Bifidobacterium longum subsp. infantis ATCC 15697 | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
monomer | 1 * 42500, SDS-PAGE | Bifidobacterium longum |
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 |
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 | Comment | Organism |
---|---|---|
hexosamine kinase | - |
Bifidobacterium longum subsp. infantis |
hexosamine kinase | - |
Bifidobacterium longum |
N-acetylhexosamine 1-phosphate kinase | - |
Bifidobacterium longum subsp. infantis |
N-acetylhexosamine 1-phosphate kinase | - |
Bifidobacterium longum |
NahK | - |
Bifidobacterium longum subsp. infantis |
NahK | - |
Bifidobacterium longum |
NahKATCC15697 | - |
Bifidobacterium longum subsp. infantis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
37 | - |
assay at | Bifidobacterium longum subsp. infantis |
37 | - |
assay at | Bifidobacterium longum |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
assay at | Bifidobacterium longum subsp. infantis |
8 | - |
assay at | Bifidobacterium longum |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | ATP/ADP binding structures, overview | Bifidobacterium longum subsp. infantis | |
ATP | ATP/ADP binding structures, overview | Bifidobacterium longum |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the sugar kinase-Hsp70-actin superfamily | Bifidobacterium longum subsp. infantis |
evolution | the enzyme belongs to the sugar kinase-Hsp70-actin superfamily | Bifidobacterium longum |
metabolism | the enzyme is part of the LNB/GNB pathway in bifidobacteria, overview | Bifidobacterium longum subsp. infantis |
metabolism | the enzyme is part of the LNB/GNB pathway in bifidobacteria, overview | Bifidobacterium longum |