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Literature summary extracted from

  • Lin, M.; Chi, M.; Chen, Y.; Wang, T.; Lo, H.; Lin, L.
    Site-directed mutagenesis of a conserved Asn450 residue of Bacillus licheniformis gamma-glutamyltranspeptidase (2016), Int. J. Biol. Macromol., 91, 416-425 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.3.2.2 gene ggt, sequence comparisons, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain M15 Bacillus licheniformis

Protein Variants

EC Number Protein Variants Comment Organism
2.3.2.2 additional information molecular modeling of mutant enzymes using the X-ray crystal structures of BlGGT (PDB ID 4OTT) and T399A-BlGGT (PDB ID 4Y23) as templates. GdnHCl-induced denaturation of BlGGT and its variants, overview Bacillus licheniformis
2.3.2.2 N450A site-directed mutagenesis, the mutant shows about 4.7fold increased catalytic efficiency compared to wild-type Bacillus licheniformis
2.3.2.2 N450D site-directed mutagenesis, the mutant shows about 8fold increased catalytic efficiency compared to wild-type Bacillus licheniformis
2.3.2.2 N450K site-directed mutagenesis, N450K exhibits 81% increase in KM and 44.3% decrease in kcat compared to wild-type, leading to a profound reduction in its catalytic efficiency Bacillus licheniformis
2.3.2.2 N450R site-directed mutagenesis, the mutant shows a significant reduction in the catalytic activity compared to wild-type Bacillus licheniformis
2.3.2.2 T399A site-directed mutagenesis, mutant structure analysis and comparison Bacillus licheniformis

General Stability

EC Number General Stability Organism
2.3.2.2 GdnHCl-induced denaturation of BlGGT and its variants, overview Bacillus licheniformis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.3.2.2 0.42
-
 L-gamma-glutamyl-4-nitroanilide recombinant wild-type enzyme, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 0.58
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450D, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 0.58
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450Q, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 0.69
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450A, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 0.76
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450K, pH 9.0, 40°C Bacillus licheniformis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.3.2.2 a (5-L-glutamyl)-peptide + an amino acid Bacillus licheniformis
-
 a peptide + a 5-L-glutamyl amino acid
-
 ?
2.3.2.2 a (5-L-glutamyl)-peptide + an amino acid Bacillus licheniformis DSM 13
-
 a peptide + a 5-L-glutamyl amino acid
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.3.2.2 Bacillus licheniformis Q62WE3
-
-
2.3.2.2 Bacillus licheniformis DSM 13 Q62WE3
-
-

Posttranslational Modification

EC Number Posttranslational Modification Comment Organism
2.3.2.2 proteolytic modification the enzyme is synthetized as single-chain precursor, and then self-processes to form the mature enzyme. Analysis of autocatalytic processing of recombinant mutant enzymes expressed in Escherichia coli strain M15 Bacillus licheniformis

Purification (Commentary)

EC Number Purification (Comment) Organism
2.3.2.2 recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain M15 by nickel affinity chromatography Bacillus licheniformis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.3.2.2 a (5-L-glutamyl)-peptide + an amino acid
-
 Bacillus licheniformis a peptide + a 5-L-glutamyl amino acid
-
 ?
2.3.2.2 a (5-L-glutamyl)-peptide + an amino acid the binding of L-glutamate occurs in a concave site lined by residues Arg109,Thr399, Glu438, Asp441, Ser460, Ser461, Gly481, and Gly482 Bacillus licheniformis a peptide + a 5-L-glutamyl amino acid
-
 ?
2.3.2.2 a (5-L-glutamyl)-peptide + an amino acid
-
 Bacillus licheniformis DSM 13 a peptide + a 5-L-glutamyl amino acid
-
 ?
2.3.2.2 a (5-L-glutamyl)-peptide + an amino acid the binding of L-glutamate occurs in a concave site lined by residues Arg109,Thr399, Glu438, Asp441, Ser460, Ser461, Gly481, and Gly482 Bacillus licheniformis DSM 13 a peptide + a 5-L-glutamyl amino acid
-
 ?
2.3.2.2 L-gamma-glutamyl-4-nitroanilide + glycylglycine
-
 Bacillus licheniformis 4-nitroaniline + 5-L-glutamyl-glycylglycine
-
 ?
2.3.2.2 L-gamma-glutamyl-4-nitroanilide + glycylglycine
-
 Bacillus licheniformis DSM 13 4-nitroaniline + 5-L-glutamyl-glycylglycine
-
 ?

Subunits

EC Number Subunits Comment Organism
2.3.2.2 heterodimer in the crystal structure of BlGGT, the large subunit contains a globular domain consisting of 14 alpha-helices, six small 310 helices and 11 beta-strands, and the small subunit comprises of 3 alpha-helices, two small 310 helices and 11 beta-strands. Furthermore, both subunits provide strands to a nearly flat beta-sheet that constitutes the core of the heterodimeric arrangement. Unlike their structural consistency, the primary structure of GGT enzymes displays a great degree of variability Bacillus licheniformis

Synonyms

EC Number Synonyms Comment Organism
2.3.2.2 BlGGT
-
 Bacillus licheniformis
2.3.2.2 gamma-glutamyltranspeptidase
-
 Bacillus licheniformis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.3.2.2 40
-
-
 Bacillus licheniformis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.3.2.2 8.93
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450K, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 16.04
-
 L-gamma-glutamyl-4-nitroanilide recombinant wild-type enzyme, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 21.74
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450Q, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 123.65
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450A, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 175.52
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450D, pH 9.0, 40°C Bacillus licheniformis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.3.2.2 9
-
-
 Bacillus licheniformis

General Information

EC Number General Information Comment Organism
2.3.2.2 evolution Bacillus licheniformis gamma-glutamyltranspeptidase (BlGGT) belongs to N-terminal nucleophile hydrolase superfamily in which all inclusive members are synthetized as single-chain precursors, and then self-processed to form mature enzymes Bacillus licheniformis
2.3.2.2 additional information the binding of L-glutamate occurs in a concave site lined by residues Arg109,Thr399, Glu438, Asp441, Ser460, Ser461, Gly481, and Gly482. Asn450 interacts with Asp441 by a hydrogenbond and consequently binds with Arg109 and Glu438 through the hydrogen bond network to hold the substrate in the properposition, suggesting that this residue can be also important for the catalytic function of BlGGT. The putative active residue is Thr399. Superimposition of the catalytic environment of wild-type and mutant enzymes, overview Bacillus licheniformis

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.3.2.2 11.75
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450K, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 37.48
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450Q, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 38.19
-
 L-gamma-glutamyl-4-nitroanilide recombinant wild-type enzyme, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 179.02
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450A, pH 9.0, 40°C Bacillus licheniformis
2.3.2.2 302.62
-
 L-gamma-glutamyl-4-nitroanilide recombinant mutant N450D, pH 9.0, 40°C Bacillus licheniformis