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 (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 | 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 | 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 | Comment | Organism | Structure |
---|---|---|---|
Mg2+ | required | Maricaulis maris |
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 | UniProt | Comment | Textmining |
---|---|---|---|
Maricaulis maris | Q0ASS9 | - |
- |
Maricaulis maris MCS10 | Q0ASS9 | - |
- |
Xanthomonas campestris | A0A0H2X8L7 | pv. campestris | - |
Xanthomonas campestris 8004 | A0A0H2X8L7 | pv. campestris | - |
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 | 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 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 | 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 | 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 Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Maricaulis maris |
30 | - |
assay at | Xanthomonas campestris |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8.5 | - |
assay at | Maricaulis maris |
8.5 | - |
assay at | Xanthomonas campestris |
Cofactor | Comment | Organism | Structure |
---|---|---|---|
ATP | - |
Maricaulis maris | |
ATP | - |
Xanthomonas campestris |
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 |