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Literature summary 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 .
    View publication on PubMedView publication on EuropePMC

Application

EC Number Application Comment Organism
3.5.1.25 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
3.5.1.25 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)

EC Number Cloned (Comment) Organism
3.5.1.25 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
3.5.1.25 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)

EC Number Crystallization (Comment) Organism
3.5.1.25 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

EC Number Protein Variants Comment Organism
3.5.1.25 D267A site-directed mutagenesis, inactive mutant Mycolicibacterium smegmatis
3.5.1.25 D272A site-directed mutagenesis, inactive mutant Mycobacterium marinum
3.5.1.25 E127A site-directed mutagenesis, inactive mutant Mycobacterium marinum
3.5.1.25 H139A site-directed mutagenesis, inactive mutant Mycobacterium marinum
3.5.1.25 H249A site-directed mutagenesis, inactive mutant Mycobacterium marinum
3.5.1.25 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
3.5.1.25 R225A site-directed mutagenesis, inactive mutant Mycobacterium marinum

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.25 1,10-phenanthroline results in unfolding and loss of catalytic activity at 10 mM Mycolicibacterium smegmatis
3.5.1.25 EDTA results in unfolding and loss of catalytic activity at 10 mM Mycolicibacterium smegmatis
3.5.1.25 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.25 additional information
-
 additional information Michaelis-Menten kinetics Mycolicibacterium smegmatis
3.5.1.25 additional information
-
 additional information Michaelis-Menten kinetics Mycobacterium marinum
3.5.1.25 3
-
 N-acetyl-D-glucosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 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
3.5.1.25 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
3.5.1.25 19.3
-
 N-acetyl-D-mannosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 20.9
-
 N-acetyl-D-glucosamine-6-sulfate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 118.5
-
 N-acetyl-D-galactosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged wild-type enzyme expressed from Mycobacterium smegmatis Mycolicibacterium smegmatis
3.5.1.25 145.4
-
 N-acetyl-D-galactosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
3.5.1.25 1,10-phenanthroline results in unfolding and loss of catalytic activity at 10 mM Mycobacterium marinum
3.5.1.25 EDTA results in unfolding and loss of catalytic activity at 10 mM Mycobacterium marinum
3.5.1.25 Fe2+ the protein contains predominantly more zinc in a 12:1 zinc:iron ratio Mycolicibacterium smegmatis
3.5.1.25 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
3.5.1.25 additional information structure of the metal-binding site in MSNagA, overview Mycolicibacterium smegmatis
3.5.1.25 Zn2+ the protein contains predominantly more zinc in a 12:1 zinc:iron ratio Mycolicibacterium smegmatis

Natural Substrates/ Products (Substrates)

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

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.25 Mycobacterium marinum A0A3E2N0G4
-
-
3.5.1.25 Mycolicibacterium smegmatis A0A8B4QF55
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.25 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli by cobalt affinity and anion exchange chromatography, followed by gel filtration Mycolicibacterium smegmatis
3.5.1.25 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)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.25 additional information no catalytic activity toward GlcNAc, GlcNGc6P, and MurNAc6P Mycobacterium marinum ?
-
 ?
3.5.1.25 additional information no catalytic activity toward N-acetyl-D-glucosamine Mycolicibacterium smegmatis ?
-
 ?
3.5.1.25 N-acetyl-D-galactosamine-6-phosphate + H2O very low activity Mycobacterium marinum D-galactosamine 6-phosphate + acetate
-
 ?
3.5.1.25 N-acetyl-D-galactosamine-6-phosphate + H2O low activity Mycolicibacterium smegmatis D-galactosamine 6-phosphate + acetate
-
 ?
3.5.1.25 N-acetyl-D-glucosamine-6-phosphate + H2O
-
 Mycolicibacterium smegmatis D-glucosamine 6-phosphate + acetate
-
 ?
3.5.1.25 N-acetyl-D-glucosamine-6-phosphate + H2O
-
 Mycobacterium marinum D-glucosamine 6-phosphate + acetate
-
 ?
3.5.1.25 N-acetyl-D-glucosamine-6-phosphate + H2O preferred substrate Mycobacterium marinum D-glucosamine 6-phosphate + acetate
-
 ?
3.5.1.25 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
-
 ?
3.5.1.25 N-acetyl-D-glucosamine-6-sulfate + H2O
-
 Mycolicibacterium smegmatis D-glucosamine 6-sulfate + acetate
-
 ?
3.5.1.25 N-acetyl-D-glucosamine-6-sulfate + H2O
-
 Mycobacterium marinum D-glucosamine 6-sulfate + acetate
-
 ?
3.5.1.25 N-acetyl-D-glucosamine-6-sulfate + H2O low activity Mycobacterium marinum D-glucosamine 6-sulfate + acetate
-
 ?
3.5.1.25 N-acetyl-D-mannosamine-6-phosphate + H2O low activity Mycolicibacterium smegmatis D-mannosamine 6-phosphate + acetate
-
 ?
3.5.1.25 N-acetyl-D-mannosamine-6-phosphate + H2O low activity Mycobacterium marinum D-mannosamine 6-phosphate + acetate
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.25 More enzyme structure comparisons, overview Mycolicibacterium smegmatis

Synonyms

EC Number Synonyms Comment Organism
3.5.1.25 MMNagA
-
 Mycobacterium marinum
3.5.1.25 MSNagA
-
 Mycolicibacterium smegmatis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.25 37
-
 assay at Mycolicibacterium smegmatis
3.5.1.25 37
-
 assay at Mycobacterium marinum

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.25 3 6 N-acetyl-D-glucosamine-6-sulfate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 5.4
-
 N-acetyl-D-galactosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 18.2
-
 N-acetyl-D-mannosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 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
3.5.1.25 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
3.5.1.25 36.8
-
 N-acetyl-D-glucosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 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

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.25 7
-
 assay at Mycolicibacterium smegmatis
3.5.1.25 7
-
 assay at Mycobacterium marinum

pH Range

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

General Information

EC Number General Information Comment Organism
3.5.1.25 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
3.5.1.25 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
3.5.1.25 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]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.1.25 0.037
-
 N-acetyl-D-galactosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 0.943
-
 N-acetyl-D-mannosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 1.72
-
 N-acetyl-D-glucosamine-6-sulfate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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
3.5.1.25 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
3.5.1.25 12.27
-
 N-acetyl-D-glucosamine-6-phosphate pH 7.0, 37°C, recombinant His6-tagged enzyme Mycobacterium marinum
3.5.1.25 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