Part:BBa_K1216003

beta-N-Acetylglucosaminidase (nagZ) from Escherichia Coli

nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli^[1].

3D representation of the β-N-Acetylglucosaminidase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=4GVG RCSB]

Usage and Biology

β-N-Acetylglucosaminidase can be used as a reporter enzyme in a histochemical reaction with substrates like 5-Bromo-4-Chloro-3-Indolyl-N-Acetyl-β-D-Glucosaminide, which will after hydrolysis release a blue chromophore^[2].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 25
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 76
  

Characterization

The final construct was sequenced.

Colorimetric response

Figure 1. Liquid cultures from E.Coli overexpressing NagZ after reacting with pNP-GlcNac, magenta-GlcNAc and X-GlcNAc. The negative control shows a liquid culture without substrate added.

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used NagZ in their project as reporter enzyme. To test the functionality of the enzyme, a liquid culture of E.Coli overexpressing NagZ was incubated with its chromogenic substrates magenta-GlcNac (produces magenta color), pNP-GlcNac (produces yellow color) and X-GlcNac (produces blue color).

Figure 2. Enzymatic reaction of NagZ with X-GlcNAc (1), magenta-GlcNAc (2) or pNP-GlcNac (3).

Cell lysate would have been tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unfortunately, this did not work until the wiki freeze.

Figure 4. Enzymatic reaction of NagZ with 4-MU-N-acetyl-β-D-glucosaminide.

Hydrolase	Substrate	Color, Absorption λmax	Stock	Liquid culture	Colonies	Response time
NagZ	4-Nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-GluNAc)	Yellow, 405 nm	15 mM in H2O	0.01 mM	15 mM	~ 1 minute
	5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (Magenta GluNAc)	Magenta, 565 nm	0.1 M in DMSO	1 mM, supplemented with BSA	50 mM, with BSA added	~ 15 minutes
	5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (X-GluNAc)	Blue, 615 nm	0.1 M in DMSO	1 mM, supplemented with BSA	50 mM, with BSA added	~ 15 minutes

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Crosstalk

To ensure specificity of the enzyme-substrate pairs used in Colisweeper, a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.

Figure 6. Liquid cultures of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing Aes, GusA, NagZ or none in a 96-well plate, with substrates indicated on the left added horizontally.

This crosstalk test was done in a 96-well plate, each well containing 200 μl from liquid cultures of our ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing either Aes, GusA, NagZ or none, each distributed among the column-wells of the plate. Horizontally, the chromogenic substrates were pipetted to the liquid cultures in the same order as their corresponding hydrolase. If specificity of the chosen enzyme-substrates pairs were given, we would expect an output as shown in the figure below (Figure 5). As Figure 6 shows, the overexpressed hydrolases cleave only the substrates they were expected to.

Figure 5. Expected outcome. Added substrates should be specifically cleaved by their hydrolases.

References

1. Cheng et al. (2000). Molecular Characterization of the b-N-Acetylglucosaminidase of Escherichia coli and Its Role in Cell Wall Recycling. Journal of Bacteriology. 182: 4836-4840.
2. [http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]