Recombinant E. coli N-Acetyl-D-Glucosamine Kinase/NAGK, CF 25 UG

• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

• Endotoxin Level

  <0.01 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to phosphorylate N-acetyl-D-glucosamine. The specific activity is >10,000 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Met1-Asp303 with C-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Met1

• Predicted Molecular Mass

  34 kDa

• SDS-PAGE

  37-38 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM HEPES, 150 mM NaCl, 10 mM MgCl₂, 10 mM CaCl₂, pH 7.0

• Recombinant E.coli N‑Acetyl‑D‑Glucosamine Kinase/NAGK (rE.coli NAGK) (Catalog # 8020-GK)

• Adenosine triphosphate (ATP) (Sigma, Catalog # A7699), 10 mM stock in deionized water

• N-acetyl-alpha -D-glucosamine (GlcNAc)  (Calbiochem, Catalog # 1079), 1 M stock in deionized water

• Universal Kinase Activity Kit (Catalog # )

• 96-well Clear Plate (Costar, Catalog # 92592)

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Universal Kinase Activity Kit by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock.

2. Prepare standard curve by performing six one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.

3. Prepare a reaction mixture composed of 0.5 mM ATP and 12.5 mM GlcNAc in Assay Buffer.

4. Dilute Coupling Phosphatase 4 (supplied in kit) to 10 µg/mL in Assay Buffer.

5. Dilute rE.coli NAGK to 1 µg/mL in Assay Buffer.

6. Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

7. Load 20 µL of the 1 µg/mL rE.coli NAGK into the plate. Include a control containing 20 µL of Assay Buffer.

8. Add 10 µL of 10 µg/mL Coupling Phosphatase 4 to the wells, excluding the standard curve.

9. Add 20 µL of reaction mixture to the wells, excluding the standard curve.

10. Cover the plate with a plate sealer and incubate at room temperature for 10 minutes.

11. Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.

12. Add 100 µL of deionized water to all wells. Mix briefly.

13. Add 30 µL of the Malachite Green Reagent B to all wells.  Mix and incubate for 20 minutes at room temperature.

14. Read plate at 620 nm (absorbance) in endpoint mode.

15. Calculate specific activity:

     *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for control.
     ** Under these conditions, the coupling rate is 0.475.

Per Reaction:

• rE.coli NAGK: 0.020 µg

• Coupling Phosphatase 4: 0.1 ug

• ATP: 0.2 mM

• GlcNAc: 5 mM

Background: N-Acetyl-D-Glucosamine Kinase/NAGK

N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) are repeating sugar units of peptidoglycan, the major component of bacterial cell wall structure and a drug target of various antibiotics including penicillin (1). Recently, interest has been generated regarding cell wall peptidoglycan catabolism, because as much as 50% of the peptidoglycan is turned over in one generation of bacterial growth (2). N-acetylglycosamine kinase (nagK) is a key enzyme for the recycling of GlcNAc in   E. coli (3). Due to its high activity, it can be used for efficient conversion of GlcNAc to GlcNAc-6-phosphate. The enzyme is assayed using a phosphatase-coupled kinase assay (4).

• References:

1. Plumbridge, J. (2009). J. Bacteriol. 191:5641.

2. Reith, J. et al (2011). J. Bacteriol. 193:5386.

3. Uehara, T. and Park, J.T.(2011) J. Bacteriol. 186:7273.

4. Wu, Z.L. (2011) PLoS One 6:e23172.

• Long Name:

  N-Acetyl-D-Glucosamine Kinase

• Entrez Gene IDs:

  55577 (Human); 56174 (Mouse); 297393 (Rat); 945664 (E. coli)

• Alternate Names:

  EC 2.7.1.59; GlcNAc Kinase; GNK; GNKGlcNAc kinase; N-acetyl-D-glucosamine kinase; N-Acetylglucosamine Kinase; N-acetylglucosamine kinaseHSA242910; NAGK