Activating Compound | Comment | Organism | Structure |
---|---|---|---|
ATP | ATP is an allosteric activator of the epimerase, the binding of the nucleotide is crucial for its catalytic properties, binding kinetics of wild-type and mutant enzymes, overview | Trichormus variabilis |
Cloned (Comment) | Organism |
---|---|
recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain NovaBlue (DE3) | Trichormus variabilis |
Protein Variants | Comment | Organism |
---|---|---|
K151V | site-directed mutagenesis, the mutation within the ATP-binding site leads to biphasic enzyme inactivation in the presence of 400 mM pyruvate | Trichormus variabilis |
K155A | site-directed mutagenesis, the mutation within the ATP-binding site leads to biphasic enzyme inactivation in the presence of 400 mM pyruvate | Trichormus variabilis |
K157L | site-directed mutagenesis, the mutation within the ATP-binding site leads to biphasic enzyme inactivation in the presence of 400 mM pyruvate | Trichormus variabilis |
K160I | site-directed mutagenesis, within the ATP-binding site, the mutant shows no inactivation by pyruvate, in contrast to the wild-type enzyme, but significantly impaired kinetic parameters compared to wild-type | Trichormus variabilis |
K160L | site-directed mutagenesis, within the ATP-binding site, the mutant shows no inactivation by pyruvate, in contrast to the wild-type enzyme, but significantly impaired kinetic parameters compared to wild-type | Trichormus variabilis |
K160N | site-directed mutagenesis, within the ATP-binding site, the mutant shows no inactivation by pyruvate, in contrast to the wild-type enzyme, but significantly impaired kinetic parameters compared to wild-type | Trichormus variabilis |
K160P | site-directed mutagenesis, within the ATP-binding site | Trichormus variabilis |
additional information | two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-D-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE) in combination with an N-acetylneuraminate lyase (NAL) from Escherichia coli. AvaAGE epimerizes N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-mannosamine (ManNAc), which then reacts with pyruvate in a NAL-catalyzed aldol condensation to form Neu5Ac. AvaAGE is inactivated by high pyruvate concentrations, which are used to push the NAL reaction toward the product side. A biphasic inactivation is observed in the presence of 50-800 mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis reveals that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE | Trichormus variabilis |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
N-acetylneuraminic acid | Neu5Ac | Trichormus variabilis | |
pyruvate | enzyme AvaAGE is inactivated by high pyruvate concentrations. A biphasic inactivation is observed in the presence of 50-800 mM pyruvate resulting in activity losses of the AvaAGE of up to 60% within the first hour. Site-directed mutagenesis reveals that pyruvate modifies one of the four lysine residues in the ATP-binding site of AvaAGE | Trichormus variabilis |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
9.5 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K155A | Trichormus variabilis | |
16.1 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K157L | Trichormus variabilis | |
16.3 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme | Trichormus variabilis | |
16.7 | - |
N-acetyl-D-mannosamine | pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme | Trichormus variabilis | |
30.1 | - |
N-acetyl-D-mannosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K157L | Trichormus variabilis | |
31.6 | - |
N-acetyl-D-mannosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K151V | Trichormus variabilis | |
44.2 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K151V | Trichormus variabilis | |
67.9 | - |
N-acetyl-D-mannosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K155A | Trichormus variabilis | |
129 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K160P | Trichormus variabilis | |
211 | - |
N-acetyl-D-mannosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K160P | Trichormus variabilis |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-Acyl-D-glucosamine | Trichormus variabilis | - |
N-Acyl-D-mannosamine | - |
r |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Trichormus variabilis | Q3M763 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain NovaBlue (DE3) by nickel affinity chromatography and dialysis | Trichormus variabilis |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | two-step enzymatic synthesis of N-acetylneuraminic acid (Neu5Ac) using an N-acyl-d-glucosamine 2-epimerase from Anabaena variabilis ATCC 29413 (AvaAGE)in combination with an N-acetylneuraminate lyase (NAL) expressed from Escherichia coli | Trichormus variabilis | ? | - |
? | |
N-acetyl-D-glucosamine | - |
Trichormus variabilis | N-acetyl-D-mannosamine | - |
r | |
N-acetyl-D-mannosamine | - |
Trichormus variabilis | N-acetyl-D-glucosamine | - |
r | |
N-Acyl-D-glucosamine | - |
Trichormus variabilis | N-Acyl-D-mannosamine | - |
r |
Synonyms | Comment | Organism |
---|---|---|
AvaAGE | - |
Trichormus variabilis |
N-acyl-D-glucosamine 2-epimerase | - |
Trichormus variabilis |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
30 | - |
assay at | Trichormus variabilis |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
8.4 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K157L | Trichormus variabilis | |
9.4 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K155A | Trichormus variabilis | |
16.1 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K151V | Trichormus variabilis | |
22.4 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K160P | Trichormus variabilis | |
44.4 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme | Trichormus variabilis |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Trichormus variabilis |
Ki Value [mM] | Ki Value maximum [mM] | Inhibitor | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | inactivation kinetics by pyruvate of wild-type and mutant enzymes, overview | Trichormus variabilis | |
10.1 | - |
N-acetylneuraminic acid | pH 7.5, 30°C, recombinant His6-tagged mutant K151V | Trichormus variabilis | |
14.3 | - |
N-acetylneuraminic acid | pH 7.5, 30°C, recombinant His6-tagged mutant K155A | Trichormus variabilis | |
15.3 | - |
N-acetylneuraminic acid | pH 7.5, 30°C, recombinant His6-tagged mutant K160P | Trichormus variabilis | |
23.3 | - |
pyruvate | pH 7.5, 30°C, recombinant His6-tagged mutant K160P | Trichormus variabilis | |
35 | - |
pyruvate | pH 7.5, 30°C, recombinant His6-tagged mutant K151V | Trichormus variabilis | |
35.1 | - |
N-acetylneuraminic acid | pH 7.5, 30°C, recombinant His6-tagged mutant K157L | Trichormus variabilis | |
38.5 | - |
pyruvate | pH 7.5, 30°C, recombinant His6-tagged mutant K155A | Trichormus variabilis | |
38.7 | - |
N-acetylneuraminic acid | pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme | Trichormus variabilis | |
69.1 | - |
pyruvate | pH 7.5, 30°C, recombinant His6-tagged mutant K157L | Trichormus variabilis | |
412 | - |
pyruvate | pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme | Trichormus variabilis |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.17 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K160P | Trichormus variabilis | |
0.36 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K151V | Trichormus variabilis | |
0.52 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K157L | Trichormus variabilis | |
0.98 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged mutant K155A | Trichormus variabilis | |
2.72 | - |
N-acetyl-D-glucosamine | pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme | Trichormus variabilis |