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Literature summary extracted from

  • Klermund, L.; Riederer, A.; Hunger, A.; Castiglione, K.
    Protein engineering of a bacterial N-acyl-D-glucosamine 2-epimerase for improved stability under process conditions (2016), Enzyme Microb. Technol., 87-88, 70-78 .
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
5.1.3.8 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(Commentary)

EC Number Cloned (Comment) Organism
5.1.3.8 recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain NovaBlue (DE3) Trichormus variabilis

Protein Variants

EC Number Protein Variants Comment Organism
5.1.3.8 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
5.1.3.8 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
5.1.3.8 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
5.1.3.8 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
5.1.3.8 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
5.1.3.8 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
5.1.3.8 K160P site-directed mutagenesis, within the ATP-binding site Trichormus variabilis
5.1.3.8 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

EC Number Inhibitors Comment Organism Structure
5.1.3.8 N-acetylneuraminic acid Neu5Ac Trichormus variabilis
5.1.3.8 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]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
5.1.3.8 9.5
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K155A Trichormus variabilis
5.1.3.8 16.1
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K157L Trichormus variabilis
5.1.3.8 16.3
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme Trichormus variabilis
5.1.3.8 16.7
-
N-acetyl-D-mannosamine pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme Trichormus variabilis
5.1.3.8 30.1
-
N-acetyl-D-mannosamine pH 7.5, 30°C, recombinant His6-tagged mutant K157L Trichormus variabilis
5.1.3.8 31.6
-
N-acetyl-D-mannosamine pH 7.5, 30°C, recombinant His6-tagged mutant K151V Trichormus variabilis
5.1.3.8 44.2
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K151V Trichormus variabilis
5.1.3.8 67.9
-
N-acetyl-D-mannosamine pH 7.5, 30°C, recombinant His6-tagged mutant K155A Trichormus variabilis
5.1.3.8 129
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K160P Trichormus variabilis
5.1.3.8 211
-
N-acetyl-D-mannosamine pH 7.5, 30°C, recombinant His6-tagged mutant K160P Trichormus variabilis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
5.1.3.8 N-Acyl-D-glucosamine Trichormus variabilis
-
N-Acyl-D-mannosamine
-
r

Organism

EC Number Organism UniProt Comment Textmining
5.1.3.8 Trichormus variabilis Q3M763
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
5.1.3.8 recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain NovaBlue (DE3) by nickel affinity chromatography and dialysis Trichormus variabilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
5.1.3.8 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 ?
-
?
5.1.3.8 N-acetyl-D-glucosamine
-
Trichormus variabilis N-acetyl-D-mannosamine
-
r
5.1.3.8 N-acetyl-D-mannosamine
-
Trichormus variabilis N-acetyl-D-glucosamine
-
r
5.1.3.8 N-Acyl-D-glucosamine
-
Trichormus variabilis N-Acyl-D-mannosamine
-
r

Synonyms

EC Number Synonyms Comment Organism
5.1.3.8 AvaAGE
-
Trichormus variabilis
5.1.3.8 N-acyl-D-glucosamine 2-epimerase
-
Trichormus variabilis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
5.1.3.8 30
-
assay at Trichormus variabilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
5.1.3.8 8.4
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K157L Trichormus variabilis
5.1.3.8 9.4
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K155A Trichormus variabilis
5.1.3.8 16.1
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K151V Trichormus variabilis
5.1.3.8 22.4
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K160P Trichormus variabilis
5.1.3.8 44.4
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme Trichormus variabilis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
5.1.3.8 7.5
-
assay at Trichormus variabilis

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
5.1.3.8 additional information
-
additional information inactivation kinetics by pyruvate of wild-type and mutant enzymes, overview Trichormus variabilis
5.1.3.8 10.1
-
N-acetylneuraminic acid pH 7.5, 30°C, recombinant His6-tagged mutant K151V Trichormus variabilis
5.1.3.8 14.3
-
N-acetylneuraminic acid pH 7.5, 30°C, recombinant His6-tagged mutant K155A Trichormus variabilis
5.1.3.8 15.3
-
N-acetylneuraminic acid pH 7.5, 30°C, recombinant His6-tagged mutant K160P Trichormus variabilis
5.1.3.8 23.3
-
pyruvate pH 7.5, 30°C, recombinant His6-tagged mutant K160P Trichormus variabilis
5.1.3.8 35
-
pyruvate pH 7.5, 30°C, recombinant His6-tagged mutant K151V Trichormus variabilis
5.1.3.8 35.1
-
N-acetylneuraminic acid pH 7.5, 30°C, recombinant His6-tagged mutant K157L Trichormus variabilis
5.1.3.8 38.5
-
pyruvate pH 7.5, 30°C, recombinant His6-tagged mutant K155A Trichormus variabilis
5.1.3.8 38.7
-
N-acetylneuraminic acid pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme Trichormus variabilis
5.1.3.8 69.1
-
pyruvate pH 7.5, 30°C, recombinant His6-tagged mutant K157L Trichormus variabilis
5.1.3.8 412
-
pyruvate pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme Trichormus variabilis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
5.1.3.8 0.17
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K160P Trichormus variabilis
5.1.3.8 0.36
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K151V Trichormus variabilis
5.1.3.8 0.52
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K157L Trichormus variabilis
5.1.3.8 0.98
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged mutant K155A Trichormus variabilis
5.1.3.8 2.72
-
N-acetyl-D-glucosamine pH 7.5, 30°C, recombinant His6-tagged wild-type enzyme Trichormus variabilis