Information on EC 2.7.1.162 - N-acetylhexosamine 1-kinase

lnpB

N-acetylhexosamine 1-kinase

N-acetylhexosamine 1-phosphate kinase

N-acetylhexosamine kinase

-

NahK

7 entries

NahKATCC15697

Bifidobacterium longum subsp. infantis

NahK_15697

-

726089

ATP + N-acetyl-D-hexosamine = ADP + N-acetyl-alpha-D-hexosamine 1-phosphate

9 entries

Phosphorylation

ATP:N-acetyl-D-hexosamine 1-phosphotransferase

This enzyme is involved in the lacto-N-biose I/galacto-N-biose degradation pathway in the probiotic bacterium Bifidobacterium longum. Differs from EC 2.7.1.157, N-acetylgalactosamine kinase, as it can phosphorylate both N-acetylgalactosamine and N-acetylglucosamine at similar rates. Also has some activity with N-acetyl-D-mannosamine, D-talose and D-mannose as substrate. ATP can be replaced by GTP or ITP but with decreased enzyme activity. Requires a divalent cation, with Mg2+ resulting in by far the greatest stimulation of enzyme activity.

959459-09-7

protein sequence

ATP + 2-(acetylamino)-2-deoxy-D-allopyranose

ADP + 2-(acetylamino)-2-deoxy-D-allopyranose 1-phosphate

22% phosphorylation yield

-

?

ATP + 2-deoxy-2-azido-D-mannose

ADP + 2-deoxy-2-azido-alpha-D-mannose 1-phosphate

ATP + 2-deoxy-2-fluoro-D-mannose

ADP + 2-deoxy-2-fluoro-alpha-D-mannose 1-phosphate

ATP + 2-deoxy-D-glucose

ADP + 2-deoxy-alpha-D-glucose 1-phosphate

ATP + 4- deoxy-N-acetyl-D-glucosamine

ADP + 4-deoxy-N-acetyl-alpha-D-glucosamine 1-phosphate

-

50% phosphorylation yield

-

?

ATP + 4-deoxy-4-azido-N-acetyl-D-galactosamine

ADP + 4-deoxy-4-azido-N-acetyl-alpha-D-galactosamine 1-phosphate

-

73% phosphorylation yield

-

?

ATP + 4-deoxy-D-mannose

ADP + 4-deoxy-alpha-D-mannose 1-phosphate

ATP + 6-deoxy-6-azido-N-acetyl-D-galactosamine

ADP + 6-deoxy-6-azido-N-acetyl-alpha-D-galactosamine 1-phosphate

-

42% phosphorylation yield

-

?

ATP + 6-deoxy-6-azido-N-acetyl-D-glucosamine

ADP + 6-deoxy-6-azido-N-acetyl-alpha-D-glucosamine 1-phosphate

ATP + 6-deoxy-6-methyl-N-acetyl-D-glucosamine

ADP + 6-deoxy-6-methyl-N-acetyl-alpha-D-glucosamine 1-phosphate

ATP + 6-deoxy-N-acetyl-D-galactosamine

ADP + 6-deoxy-N-acetyl-alpha-D-galactosamine 1-phosphate

-

37% phosphorylation yield

-

?

ATP + 6-deoxy-N-acetyl-D-glucosamine

ADP + 6-deoxy-N-acetyl-alpha-D-glucosamine 1-phosphate

75% phosphorylation yield

-

?

ATP + 6-methyl-N-acetyl-D-mannosamine

ADP + 6-methyl-N-acetyl-alpha-D-mannosamine 1-phosphate

ATP + D-galactopyranose

ADP + alpha-D-galactopyranose 1-phosphate

-

below 5% phosphorylation yield

-

?

ATP + D-galactosamine

ADP + D-galactosamine 1-phosphate

ATP + D-galactose

ADP + D-galactose 1-phosphate

ATP + D-glucosamine

ADP + D-glucosamine 1-phosphate

-

-

?

ATP + D-glucose

ADP + D-glucose 1-phosphate

low activity

-

?

ATP + D-mannosamine

ADP + alpha-D-mannosamine 1-phosphate

-

-

?

ATP + D-mannose

?

ATP + D-mannose

ADP + alpha-D-mannose 1-phosphate

ATP + D-talose

?

phosphorylation at 3% of the activity with N-acetylglucosamine

-

?

ATP + mannose

ADP + mannose 1-phosphate

-

-

?

ATP + N-acetyl-alpha-D-galactosamine

ADP + N-acetyl-alpha-D-galactosamine 1-phosphate

10 entries

ATP + N-acetyl-alpha-D-glucosamine

ADP + N-acetyl-alpha-D-glucosamine 1-phosphate

7 entries

ATP + N-acetyl-D-galactosamine

ADP + N-acetyl-alpha-D-galactosamine 1-phosphate

6 entries

ATP + N-acetyl-D-glucosamine

ADP + N-acetyl-alpha-D-glucosamine 1-phosphate

8 entries

ATP + N-acetyl-D-hexosamine

ADP + N-acetyl-alpha-D-hexosamine 1-phosphate

ATP + N-acetyl-D-mannosamine

?

phosphorylation at 15% of the activity with N-acetylglucosamine

-

?

ATP + N-acetyl-D-mannosamine

ADP + ?

-

-

?

ATP + N-acetyl-D-mannosamine

ADP + N-acetyl-alpha-D-mannosamine 1-phosphate

low activity

-

?

ATP + N-azidoacetyl-D-galactosamine

ADP + N-azidoacetyl-alpha-D-galactosamine 1-phosphate

-

65% phosphorylation yield

-

?

ATP + N-azidoacetyl-D-glucosamine

ADP + N-azidoacetyl-alpha-D-glucosamine 1-phosphate

87% phosphorylation yield

-

?

ATP + N-benzoyl-alpha-D-galactosamine

ADP + N-benzoyl-alpha-D-galactosamine 1-phosphate

ATP + N-butanoyl-D-galactosamine

ADP + N-butanoyl-alpha-D-galactosamine 1-phosphate

ATP + N-butanoyl-D-glucosamine

ADP + N-butanoyl-alpha-D-glucosamine 1-phosphate

ATP + N-phenylacetyl-D-galactosamine

ADP + N-phenylacetyl-alpha-D-galactosamine 1-phosphate

-

77% phosphorylation yield

-

?

ATP + N-phenylacetyl-D-glucosamine

ADP + N-phenylacetyl-alpha-D-glucosamine 1-phosphate

88% phosphorylation yield

-

?

ATP + N-propionyl-D-galactosamine

ADP + N-propionyl-alpha-D-galactosamine 1-phosphate

-

85% phosphorylation yield

-

?

ATP + N-propionyl-D-glucosamine

ADP + N-propionyl-alpha-D-glucosamine 1-phosphate

86% phosphorylation yield

-

?

ATP + N-trifluoroacetyl-alpha-D-galactosamine

ADP + N-trifluoroacetyl-alpha-D-galactosamine 1-phosphate

-

0.75 microM, 21.8% conversion

723238

-

?

ATP + N-trifluoroacetyl-D-glucosamine

ADP + N-trifluoroacetyl-alpha-D-glucosamine 1-phosphate

Escherichia coli

-

recombinant enzyme

739523

-

?

GTP + N-acetyl-alpha-D-glucosamine

GDP + N-acetyl-alpha-D-glucosamine 1-phosphate

GTP shows about 45% of the activity with ATP

-

?

ITP + N-acetyl-alpha-D-glucosamine

IDP + N-acetyl-alpha-D-glucosamine 1-phosphate

ITP shows about 30% of the activity with ATP

-

?

additional information

?

-

9 entries

ATP + N-acetyl-alpha-D-galactosamine

ADP + N-acetyl-alpha-D-galactosamine 1-phosphate

ATP + N-acetyl-alpha-D-glucosamine

ADP + N-acetyl-alpha-D-glucosamine 1-phosphate

ATP + N-acetyl-D-hexosamine

ADP + N-acetyl-alpha-D-hexosamine 1-phosphate

ATP

GTP

-

ITP

-

Ca2+

1 mM, 1% relative activity compared to Mg2+

Co2+

Mg2+

7 entries

MgCl2

-

10 mM are included in the assay medium

Mn2+

1 mM, 7% relative activity compared to Mg2+

Ni2+

1 mM, 4% relative activity compared to Mg2+

Zn2+

1 mM, 8% relative activity compared to Mg2+

additional information

isopropyl-1-thio-beta-D-galactoside

induction of protein expression at a final concentration of 0.5 mM

0.005 - 0.9

ATP

10 entries

0.065 - 0.09

N-acetyl-alpha-D-galactosamine

0.06 - 0.118

N-acetyl-alpha-D-glucosamine

0.35 - 3.14

N-acetyl-D-glucosamine

additional information

-

0.12 - 16.3

ATP

8 entries

0.46 - 6.08

N-acetyl-alpha-D-galactosamine

0.95 - 1.21

N-acetyl-alpha-D-glucosamine

0.11 - 13.6

N-acetyl-D-glucosamine

4.8 - 11.8

ATP

5.1 - 7.1

N-acetyl-alpha-D-galactosamine

15.8 - 18.3

N-acetyl-alpha-D-glucosamine

0.001

D-glucose, substrate donor specificity

0.002

D-mannosamine, substrate donor specificity

0.004

D-glucosamine, substrate donor specificity

0.007

2-deoxyglucose, substrate donor specificity

0.024

D-mannose, substrate donor specificity

0.049

D-talose, substrate donor specificity

0.22

N-acetyl-D-mannosamine, substrate donor specificity

0.48

ITP, substrate donor specificity

0.65

GTP, substrate donor specificity

0.89

N-acetyl-D-galactosamine, substrate donor specificity

1.48

ATP, substrate donor specificity

1.49

N-acetyl-D-glucosamine, substrate donor specificity

additional information

7 - 8

8

8.5

9

-

assay at

37

40

-

Bifidobacterium longum ATCC 55813

-

723238

-

Bifidobacterium longum subsp. infantis

Bifidobacterium longum subsp. infantis ATCC 15697

-

-

evolution

metabolism

additional information

Bifidobacterium longum subsp. infantis ATCC 15697 = JCM 1222 = DSM 20088

Bifidobacterium longum subsp. longum JCM 1217

42500

dimer

monomer

additional information

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

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 subsp. infantis

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

-

D208 A

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

D208A

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

D208A/K210A

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

D208A/K210L

site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type

D208L

site-directed mutagenesis, the mutant shows highly reduced activity and altered substrate specificity compared to the wild-type

D228A

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

I146E

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

I146E/T231E

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

K210A

K210E

site-directed mutagenesis, the mutant shows reduced highly activity and altered substrate specificity compared to the wild-type

K210L

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

N213A

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

Q48A

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

R306A

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

R306A/Y317A

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

R306E

site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type

R306E/Y317F

site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type

T231 E

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

Y317A

site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type

Y317F

site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type

D208A

-

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

0

D228A

-

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

0

K210A

-

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

0

N213A

-

site-directed mutagenesis, the mutant shows altered activity and kinetics compared to the wild-type enzyme

0

Q48A

-

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

0

R306A

-

site-directed mutagenesis, the mutant shows reduced activity and altered substrate specificity compared to the wild-type

0

R306E

-

site-directed mutagenesis, the mutant shows highly reduced activity but unaltered substrate specificity compared to the wild-type

0

Y317A

-

site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type

0

Y317F

-

site-directed mutagenesis, the mutant shows similar activity but slightly altered substrate specificity compared to the wild-type

0

5 - 9.5

50

pH 8.5, 30 min, 50% loss of activity

loses half of enzyme activity during incubation for 30 min at 50°C in 0.1 M Tris buffer (pH 8.5)

-

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

recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration

The transformant is cultivated and protein expression is induced by the addition of isopropyl-1-thio-beta-D-galactoside. The cells are harvested by centrifugation and sonicated. Cell debris is removed by centrifugation and enzyme is purified on an Ni-nitrilotriacetic acid agarose gel.

Escherichia coli strains select96 and BL21(DE3) are used as hosts for cloning and expression.

expression in Escherichia coli

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)

gene lnpB, recombinant expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)