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Literature summary extracted from

  • Zhao, G.; Haskins, N.; Jin, Z.; M Allewell, N.; Tuchman, M.; Shi, D.
    Structure of N-acetyl-L-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor L-arginine bound (2013), Biochem. Biophys. Res. Commun., 437, 585-590.
    View publication on PubMedView publication on EuropePMC

Activating Compound

EC Number Activating Compound Comment Organism Structure
2.3.1.1 L-arginine
-
 Maricaulis maris

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.1.1 expressed in Escherichia coli BL21(DE3) cells Maricaulis maris
2.7.2.8 recombinant His-tagged wild-type and mutant enzymes expression in Escherichia coli BL21(DE3) Maricaulis maris
2.7.2.8 recombinant His-tagged wild-type and mutant enzymes expression in Escherichia coli BL21(DE3) Xanthomonas campestris

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.3.1.1 N-acetyl-L-glutamate synthase/kinase in complex with L-arginine, sitting drop vapor diffusion method, using 100 mM sodium cacodylate trihydrate, pH 6.2, 25% (w/v) polypropylene glycol P400 and 200 mM magnesium chloride Maricaulis maris
2.7.2.8 purified enzyme in complex with L-arginine, sitting drop vapor diffusion method, mixing of 10 mg/ml protein in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 10% glycerol, 5 mM 2-mercaptoethanol, and 1 mM EDTA with 1 mM L-arginine, and 10 mM N-acetyl-L-glutamate for 30 min, 0.002 ml of this protein solution is mixed with 0.002 ml of reservoir solution containing 100 mM sodium cacodylate trihydrate, pH 6.2, 25% PEG P400 and 200 mM magnesium chloride, X-ray diffraction structure determination and analysis at 2.8 A resolution. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations Maricaulis maris

Protein Variants

EC Number Protein Variants Comment Organism
2.3.1.1 K356H inactive Maricaulis maris
2.3.1.1 N391Q inactive Maricaulis maris
2.3.1.1 R386K inactive Maricaulis maris
2.3.1.1 S387A the mutant shows reduced activity compared to the wild type enzyme Maricaulis maris
2.3.1.1 Y397F inactive Maricaulis maris
2.7.2.8 K356H site-directed mutagenesis, inactive mutant Maricaulis maris
2.7.2.8 K364H site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Xanthomonas campestris
2.7.2.8 N391Q site-directed mutagenesis, inactive mutant Maricaulis maris
2.7.2.8 N399Q site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Xanthomonas campestris
2.7.2.8 R386K site-directed mutagenesis, inactive mutant Maricaulis maris
2.7.2.8 R394K site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme Xanthomonas campestris
2.7.2.8 S387A site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme Maricaulis maris
2.7.2.8 S395A site-directed mutagenesis, the mutant shows slightly reduced activity compared to the wild-type enzyme Xanthomonas campestris
2.7.2.8 Y397F site-directed mutagenesis, inactive mutant Maricaulis maris
2.7.2.8 Y405F site-directed mutagenesis, inactive mutant Xanthomonas campestris

Inhibitors

EC Number Inhibitors Comment Organism Structure
2.7.2.8 L-arginine an allosteric inhibitor of mmNAGS/K, mechanism of allosteric inhibition of mmNAGS/K by L-arginine. L-Arginine is an allosteric inhibitor of NAGS/K but an allosteric activator of mammalian NAGS. In contrast to the structure of mmNAGS/K in the absence of L-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the L-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase domain is blocked by a loop from the amino acid kinase domain, as a result of a domain rotation that occurs when L-arginine binds Maricaulis maris
2.7.2.8 trichloroacetic acid complete inactivation at 30% Maricaulis maris

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.2.8 Mg2+ required Maricaulis maris

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.1.1 acetyl-CoA + L-glutamate Maricaulis maris
-
 CoA + N-acetyl-L-glutamate
-
 ?
2.3.1.1 acetyl-CoA + L-glutamate Maricaulis maris MCS10
-
 CoA + N-acetyl-L-glutamate
-
 ?
2.7.2.8 ATP + N-acetyl-L-glutamate Maricaulis maris
-
 ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 ATP + N-acetyl-L-glutamate Xanthomonas campestris
-
 ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 ATP + N-acetyl-L-glutamate Maricaulis maris MCS10
-
 ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 ATP + N-acetyl-L-glutamate Xanthomonas campestris 8004
-
 ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r

Organism

EC Number Organism UniProt Comment Textmining
2.3.1.1 Maricaulis maris Q0ASS9
-
-
2.7.2.8 Maricaulis maris Q0ASS9
-
-
2.7.2.8 Maricaulis maris MCS10 Q0ASS9
-
-
2.7.2.8 Xanthomonas campestris A0A0H2X8L7 pv. campestris
-
2.7.2.8 Xanthomonas campestris 8004 A0A0H2X8L7 pv. campestris
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.1.1 nickel affinity column chromatography and DEAE column chromatography Maricaulis maris
2.7.2.8 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3) by nickel affinity chromatography Maricaulis maris
2.7.2.8 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli BL21(DE3) by nickel affinity chromatography Xanthomonas campestris

Reaction

EC Number Reaction Comment Organism Reaction ID
2.7.2.8 ATP + N-acetyl-L-glutamate = ADP + N-acetyl-L-glutamyl 5-phosphate allosterically regulated mechanism for the enzyme from Maricaulis maris with roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism Maricaulis maris
a

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.3.1.1 4.97
-
 mutant enzyme S387A, at pH 8.5 and 30°C Maricaulis maris
2.3.1.1 6.81
-
 wild type enzyme, at pH 8.5 and 30°C Maricaulis maris
2.7.2.8 0.47
-
 purified mutant K364H enzyme, pH 8.5, 30°C Xanthomonas campestris
2.7.2.8 1.66
-
 purified mutant R394K enzyme, pH 8.5, 30°C Xanthomonas campestris
2.7.2.8 3.11
-
 purified mutant N399Q enzyme, pH 8.5, 30°C Xanthomonas campestris
2.7.2.8 4.97
-
 purified mutant S387A enzyme, pH 8.5, 30°C Maricaulis maris
2.7.2.8 6.81
-
 purified recominant wild-type enzyme, pH 8.5, 30°C Maricaulis maris
2.7.2.8 39.87
-
 purified mutant S387A enzyme, pH 8.5, 30°C Xanthomonas campestris
2.7.2.8 44.05
-
 purified recominant wild-type enzyme, pH 8.5, 30°C Xanthomonas campestris

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.1.1 acetyl-CoA + L-glutamate
-
 Maricaulis maris CoA + N-acetyl-L-glutamate
-
 ?
2.3.1.1 acetyl-CoA + L-glutamate
-
 Maricaulis maris MCS10 CoA + N-acetyl-L-glutamate
-
 ?
2.7.2.8 ATP + N-acetyl-L-glutamate
-
 Maricaulis maris ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 ATP + N-acetyl-L-glutamate
-
 Xanthomonas campestris ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 ATP + N-acetyl-L-glutamate
-
 Maricaulis maris MCS10 ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 ATP + N-acetyl-L-glutamate
-
 Xanthomonas campestris 8004 ADP + N-acetyl-L-glutamyl 5-phosphate
-
 r
2.7.2.8 additional information residues Lys364, Arg394 and Asn399 in xcNAGS/K are involved in binding of L-glutamate Xanthomonas campestris ?
-
 ?
2.7.2.8 additional information residues Lys364, Arg394 and Asn399 in xcNAGS/K are involved in binding of L-glutamate Xanthomonas campestris 8004 ?
-
 ?

Synonyms

EC Number Synonyms Comment Organism
2.3.1.1 N-acetyl-L-glutamate synthase/kinase
-
 Maricaulis maris
2.3.1.1 NAGS
-
 Maricaulis maris
2.7.2.8 mmNAGS/K
-
 Maricaulis maris
2.7.2.8 N-acetyl-L-glutamate synthase/kinase
-
 Maricaulis maris
2.7.2.8 N-acetyl-L-glutamate synthase/kinase
-
 Xanthomonas campestris
2.7.2.8 N-acetylglutamate kinase
-
 Maricaulis maris
2.7.2.8 N-acetylglutamate kinase
-
 Xanthomonas campestris
2.7.2.8 NagK
-
 Maricaulis maris
2.7.2.8 NagK
-
 Xanthomonas campestris
2.7.2.8 xcNAGS/K
-
 Xanthomonas campestris

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.2.8 30
-
 assay at Maricaulis maris
2.7.2.8 30
-
 assay at Xanthomonas campestris

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.2.8 8.5
-
 assay at Maricaulis maris
2.7.2.8 8.5
-
 assay at Xanthomonas campestris

Cofactor

EC Number Cofactor Comment Organism Structure
2.7.2.8 ATP
-
 Maricaulis maris
2.7.2.8 ATP
-
 Xanthomonas campestris

General Information

EC Number General Information Comment Organism
2.7.2.8 evolution the allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS. L-Arginine is an allosteric inhibitor of NAGS/K but an allosteric activator of mammalian NAGS Maricaulis maris
2.7.2.8 metabolism N-acetylglutamate synthase/kinase catalyzes the first two steps in L-arginine biosynthesis Xanthomonas campestris
2.7.2.8 metabolism N-acetylglutamate synthase/kinase catalyzes the first two steps in L-arginine biosynthesis. The synthase activity of mmNAGS/K is allosterically regulated by L-arginine Maricaulis maris