Literature summary for 3.5.1.25 extracted from

Ahangar, M.S.; Furze, C.M.; Guy, C.S.; Cooper, C.; Maskew, K.S.; Graham, B.; Cameron, A.D.; Fullam, E.
Structural and functional determination of homologs of the Mycobacterium tuberculosis N-acetylglucosamine-6-phosphate deacetylase (NagA) (2018), J. Biol. Chem., 293, 9770-9783 .

Search for more literature for 3.5.1.25

Application

Application	Comment	Organism
drug development	NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments	Mycolicibacterium smegmatis
drug development	NagA is a potential antitubercular drug target because it represents the key enzymatic step in the generation of essential amino-sugar precursors required for Mtb cell wall biosynthesis and also influences recycling of cell wall peptidoglycan fragments	Mycobacterium marinum

Cloned(Commentary)

Cloned (Comment)	Organism
recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli, coexpression with the Mtb GroES chaperone or pYUB1062	Mycobacterium marinum
recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli, coexpression with the Mtb GroES chaperone or pYUB1062, or overexpression in Mycobacterium smegmatis	Mycolicibacterium smegmatis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant His6-tagged enzyme MSNagA in both ligand-free form and in complex with the N-acetyl-D-glucosamine-6-phosphate substrate, X-ray diffraction structure determination and analysis at 2.6 and 2.0 A resolutions, respectively, molecular replacement	Mycolicibacterium smegmatis

Protein Variants

Protein Variants	Comment	Organism
D267A	site-directed mutagenesis, inactive mutant	Mycolicibacterium smegmatis
D272A	site-directed mutagenesis, inactive mutant	Mycobacterium marinum
E127A	site-directed mutagenesis, inactive mutant	Mycobacterium marinum
H139A	site-directed mutagenesis, inactive mutant	Mycobacterium marinum
H249A	site-directed mutagenesis, inactive mutant	Mycobacterium marinum
additional information	construction of mutant enzymes mutated to either a QXN motif to replicate the single metal-binding motif found in both the Escherichia coli and Vibrio cholerae NagA enzymes, or to AXA, resulting in highly reduced activity for the QXN mutant and complete loss of activity for the AXA mutant	Mycobacterium marinum
R225A	site-directed mutagenesis, inactive mutant	Mycobacterium marinum

Inhibitors

Inhibitors	Comment	Organism	Structure
1,10-phenanthroline	results in unfolding and loss of catalytic activity at 10 mM	Mycolicibacterium smegmatis
EDTA	results in unfolding and loss of catalytic activity at 10 mM	Mycolicibacterium smegmatis
additional information	after treatment with 10 mM 1,10-phenanthroline or 10 mM EDTA, resulting in unfolding and loss of catalytic activity, the activity cannot be restored by addition of ZnCl2 to the metal-free enzyme, indicating that the metal ions are tightly coordinated in the active site and have an essential structural role in mycobacterial NagA enzymes	Mycolicibacterium smegmatis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	Michaelis-Menten kinetics	Mycolicibacterium smegmatis
additional information	-	additional information	Michaelis-Menten kinetics	Mycobacterium marinum
3	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
3.2	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
5.2	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant wild-type His6-tagged enzyme expressed from Escherichia coli	Mycolicibacterium smegmatis
19.1	-	N-acetyl-D-mannosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
19.3	-	N-acetyl-D-mannosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
20.9	-	N-acetyl-D-glucosamine-6-sulfate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
21.4	-	N-acetyl-D-glucosamine-6-sulfate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
118.5	-	N-acetyl-D-galactosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
145.4	-	N-acetyl-D-galactosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum

Metals/Ions

Metals/Ions	Comment	Organism	Structure
1,10-phenanthroline	results in unfolding and loss of catalytic activity at 10 mM	Mycobacterium marinum
EDTA	results in unfolding and loss of catalytic activity at 10 mM	Mycobacterium marinum
Fe2+	the protein contains predominantly more zinc in a 12:1 zinc:iron ratio	Mycolicibacterium smegmatis
additional information	after treatment with 10 mM 1,10-phenanthroline or 10 mM EDTA, resulting in unfolding and loss of catalytic activity, the activity cannot be restored by addition of ZnCl2 to the metal-free enzyme, indicating that the metal ions are tightly coordinated in the active site and have an essential structural role in mycobacterial NagA enzymes	Mycobacterium marinum
additional information	structure of the metal-binding site in MSNagA, overview	Mycolicibacterium smegmatis
Zn2+	the protein contains predominantly more zinc in a 12:1 zinc:iron ratio	Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
N-acetyl-D-glucosamine-6-phosphate + H2O	Mycolicibacterium smegmatis	-	D-glucosamine 6-phosphate + acetate	-	?
N-acetyl-D-glucosamine-6-phosphate + H2O	Mycobacterium marinum	-	D-glucosamine 6-phosphate + acetate	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium marinum	A0A3E2N0G4	-	-
Mycolicibacterium smegmatis	A0A8B4QF55	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli by cobalt affinity and anion exchange chromatography, followed by gel filtration	Mycolicibacterium smegmatis
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli by cobalt affinity and anion exchange chromatography, followed by gel filtration	Mycobacterium marinum

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	no catalytic activity toward GlcNAc, GlcNGc6P, and MurNAc6P	Mycobacterium marinum	?	-	?
additional information	no catalytic activity toward N-acetyl-D-glucosamine	Mycolicibacterium smegmatis	?	-	?
N-acetyl-D-galactosamine-6-phosphate + H2O	very low activity	Mycobacterium marinum	D-galactosamine 6-phosphate + acetate	-	?
N-acetyl-D-galactosamine-6-phosphate + H2O	low activity	Mycolicibacterium smegmatis	D-galactosamine 6-phosphate + acetate	-	?
N-acetyl-D-glucosamine-6-phosphate + H2O	-	Mycolicibacterium smegmatis	D-glucosamine 6-phosphate + acetate	-	?
N-acetyl-D-glucosamine-6-phosphate + H2O	-	Mycobacterium marinum	D-glucosamine 6-phosphate + acetate	-	?
N-acetyl-D-glucosamine-6-phosphate + H2O	preferred substrate	Mycobacterium marinum	D-glucosamine 6-phosphate + acetate	-	?
N-acetyl-D-glucosamine-6-phosphate + H2O	preferred substrate, structure of the GlcNAc6P substrate-binding site in MSNagA, overview	Mycolicibacterium smegmatis	D-glucosamine 6-phosphate + acetate	-	?
N-acetyl-D-glucosamine-6-sulfate + H2O	-	Mycolicibacterium smegmatis	D-glucosamine 6-sulfate + acetate	-	?
N-acetyl-D-glucosamine-6-sulfate + H2O	-	Mycobacterium marinum	D-glucosamine 6-sulfate + acetate	-	?
N-acetyl-D-glucosamine-6-sulfate + H2O	low activity	Mycobacterium marinum	D-glucosamine 6-sulfate + acetate	-	?
N-acetyl-D-mannosamine-6-phosphate + H2O	low activity	Mycolicibacterium smegmatis	D-mannosamine 6-phosphate + acetate	-	?
N-acetyl-D-mannosamine-6-phosphate + H2O	low activity	Mycobacterium marinum	D-mannosamine 6-phosphate + acetate	-	?

Subunits

Subunits	Comment	Organism
More	enzyme structure comparisons, overview	Mycolicibacterium smegmatis

Synonyms

Synonyms	Comment	Organism
MMNagA	-	Mycobacterium marinum
MSNagA	-	Mycolicibacterium smegmatis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Mycolicibacterium smegmatis
37	-	assay at	Mycobacterium marinum

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
3	6	N-acetyl-D-glucosamine-6-sulfate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
5.4	-	N-acetyl-D-galactosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
18.2	-	N-acetyl-D-mannosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
20.7	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Escherichia coli	Mycolicibacterium smegmatis
31.1	-	N-acetyl-D-galactosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
33.2	-	N-acetyl-D-glucosamine-6-sulfate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
36.8	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
70.6	-	N-acetyl-D-mannosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
91.1	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7	-	assay at	Mycolicibacterium smegmatis
7	-	assay at	Mycobacterium marinum

pH Range

pH Minimum	pH Maximum	Comment	Organism
additional information	-	pH profile	Mycolicibacterium smegmatis
additional information	-	pH profile	Mycobacterium marinum

General Information

General Information	Comment	Organism
metabolism	the enzyme NagA catalyzes a metabolic step that connects the glycolysis pathway with the peptidoglycan biosynthesis and the synthesis of cell wall macromolecules, overview	Mycolicibacterium smegmatis
metabolism	the enzyme NagA catalyzes a metabolic step that connects the glycolysis pathway with the peptidoglycan biosynthesis and the synthesis of cell wall macromolecules, overview	Mycobacterium marinum
additional information	the GlcNAc6P complex structure discloses the precise mode of GlcNAc6P binding and the structural framework of the active site, including two divalent metals located in the alpha/beta binuclear site. Enzyme structure comparisons, overview	Mycolicibacterium smegmatis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism	Structure
0.037	-	N-acetyl-D-galactosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
0.26	-	N-acetyl-D-galactosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
0.943	-	N-acetyl-D-mannosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
1.55	-	N-acetyl-D-glucosamine-6-sulfate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
1.72	-	N-acetyl-D-glucosamine-6-sulfate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
3.7	-	N-acetyl-D-mannosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis
3.98	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Escherichia coli	Mycolicibacterium smegmatis
12.27	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged enzyme	Mycobacterium marinum
28.47	-	N-acetyl-D-glucosamine-6-phosphate	pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis	Mycolicibacterium smegmatis

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Release 2023.1 (January 2023)