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Literature summary for 4.1.1.15 extracted from

  • Liu, Q.; Cheng, H.; Ma, X.; Xu, N.; Liu, J.; Ma, Y.
    Expression, characterization and mutagenesis of a novel glutamate decarboxylase from Bacillus megaterium (2016), Biotechnol. Lett., 38, 1107-1113 .
    View publication on PubMed
Search for more literature for 4.1.1.15

Application

Application Comment Organism
synthesis the unusual high activity of BmGAD at pH 6 makes it an attractive GABA-producing candidate in industrial application Priestia megaterium

Cloned(Commentary)

Cloned (Comment) Organism
gene gad, cloned from genomic DNA, DNA and amino acid sequence determination and analysis, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha Priestia megaterium

Protein Variants

Protein Variants Comment Organism
D231R site-directed mutagenesis, the mutation leads to significant decreases in BmGAD protein levels Priestia megaterium
D38K site-directed mutagenesis Priestia megaterium
D92A site-directed mutagenesis, active site mutant, shows reduced activity at pH 5.0 compared to wild-type GAD Priestia megaterium
E179K site-directed mutagenesis, the mutant shows a slight higher activity compared to wild-type GAD in the pH range from pH 5.0-5.5, the activity of decreases sharply at values above pH 5.5 Priestia megaterium
E294R site-directed mutagenesis, the mutant increased activity of 119% compared to wild-type and shows a higher Vmax value than that of wild-type with 210 U/mg at pH 5.0 and 50°C Priestia megaterium
H467A site-directed mutagenesis, the mutant increased activity of 118% compared to wild-type and shows a higher Vmax value than that of wild-type with 180 U/mg at pH 5.0 and 50°C Priestia megaterium
additional information a constructed mutant DELTA466-467 shows reduced activity at pH 5.0 compared to wild-type GAD Priestia megaterium

Inhibitors

Inhibitors Comment Organism Structure
Ca2+ about 5% inhibition at 2 mM Priestia megaterium
Cu2+ almost complete inhibition at 2 mM Priestia megaterium
Fe2+ 50% inhibition at 2 mM Priestia megaterium
Fe3+ 85% inhibition at 2 mM Priestia megaterium
Zn2+ about 20% inhibition at 2 mM Priestia megaterium

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
8.1
-
 L-glutamate recombinant wild-type enzyme, pH 5.0, 50°C Priestia megaterium
9.2
-
 L-glutamate recombinant mutant H467A, pH 5.0, 50°C Priestia megaterium
18
-
 L-glutamate recombinant mutant E294R, pH 5.0, 50°C Priestia megaterium

Metals/Ions

Metals/Ions Comment Organism Structure
additional information Na+, Ca2+, Co2+ and Mn2+ have no effect on the activity of BmGAD at 2 mM Priestia megaterium

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-glutamate Priestia megaterium
-
 4-aminobutanoate + CO2
-
 ?
L-glutamate Priestia megaterium CICC10055
-
 4-aminobutanoate + CO2
-
 ?

Organism

Organism UniProt Comment Textmining
Priestia megaterium A0A0U3JTC9
-
-
Priestia megaterium CICC10055 A0A0U3JTC9
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration Priestia megaterium

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
59
-
 purified recombinant wild-type enzyme, pH 6.0, 50°C Priestia megaterium
148
-
 purified recombinant wild-type enzyme, pH 5.0, 50°C Priestia megaterium

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-glutamate
-
 Priestia megaterium 4-aminobutanoate + CO2
-
 ?
L-glutamate BmGAD can catalyze transformation of glutamate to GABA with a conversion rate of 28.5% (mol/mol) in 4 h at 30°C Priestia megaterium 4-aminobutanoate + CO2
-
 ?
L-glutamate
-
 Priestia megaterium CICC10055 4-aminobutanoate + CO2
-
 ?
L-glutamate BmGAD can catalyze transformation of glutamate to GABA with a conversion rate of 28.5% (mol/mol) in 4 h at 30°C Priestia megaterium CICC10055 4-aminobutanoate + CO2
-
 ?

Subunits

Subunits Comment Organism
? x * 53000-55000, recombinant His-tagged enzyme, SDS-PAGE Priestia megaterium

Synonyms

Synonyms Comment Organism
BmGAD
-
 Priestia megaterium
GAD
-
 Priestia megaterium

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
50
-
 recombinant enzyme Priestia megaterium

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
40 65 over 50% of maximal activity within this range, profile overview Priestia megaterium

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5
-
 recombinant enzyme Priestia megaterium

pH Range

pH Minimum pH Maximum Comment Organism
additional information
-
 effect of pH on enzymatic activities of recombinant wild-type and mutant BmGADs, overview Priestia megaterium
4 6.2 activity range, inactive below and above, profile overview Priestia megaterium

pH Stability

pH Stability pH Stability Maximum Comment Organism
4 7 purified recombinant BmGAD, stable at around pH 7.0, but retains only 66 % activity of its initial activity after incubation for 6 h at pH 4.0 Priestia megaterium

Cofactor

Cofactor Comment Organism Structure
pyridoxal 5'-phosphate best at 0.1 mM Priestia megaterium