Any feedback?
Please rate this page
(literature.php)
(0/150)

BRENDA support

Literature summary for 2.7.2.8 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

Cloned(Commentary)

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

Crystallization (Commentary)

Crystallization (Comment) Organism
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

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

Inhibitors

Inhibitors Comment Organism Structure
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
trichloroacetic acid complete inactivation at 30% Maricaulis maris

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Maricaulis maris

Natural Substrates/ Products (Substrates)

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

Organism

Organism UniProt Comment Textmining
Maricaulis maris Q0ASS9
-
-
Maricaulis maris MCS10 Q0ASS9
-
-
Xanthomonas campestris A0A0H2X8L7 pv. campestris
-
Xanthomonas campestris 8004 A0A0H2X8L7 pv. campestris
-

Purification (Commentary)

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

Reaction

Reaction Comment Organism Reaction ID
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

Specific Activity [micromol/min/mg]

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

Substrates and Products (Substrate)

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

Synonyms

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

Temperature Optimum [°C]

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

pH Optimum

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

Cofactor

Cofactor Comment Organism Structure
ATP
-
Maricaulis maris
ATP
-
Xanthomonas campestris

General Information

General Information Comment Organism
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
metabolism N-acetylglutamate synthase/kinase catalyzes the first two steps in L-arginine biosynthesis Xanthomonas campestris
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