Cloned (Comment) | Organism |
---|---|
gene dapE, recombinant expression of N-terminally His6-tagged enzyme, containing a TEV protease recognition site followed by the DapE catalytic domain, in an Escherichia coli strain BL21(DE3) derivative that harbors the pMAGIC plasmid encoding one rare Escherichia coli Arg tRNA (covering codons AGG/AGA) | Vibrio cholerae |
gene dapE, recombinant expression of N-terminally His6-tagged wild-type and mutant enzymes, containing a TEV protease recognition site followed by the DapE catalytic domain, in an Escherichia coli strain BL21(DE3) derivative that harbors the pMAGIC plasmid encoding one rare Escherichia coli Arg tRNA (covering codons AGG/AGA) | Haemophilus influenzae |
Crystallization (Comment) | Organism |
---|---|
purified recombinant enzyme, apoform and Zn2+-bound enzyme, using 400 nl of a precipitant solution containing 20% v/v 1,4-butanediol, 0.1 M sodium acetate, pH 4.5, and 400 nl of 19 mg/ml protein in crystallization buffer, with or without 1 mM ZnCl2, within 14 days, X-ray diffraction structure determination and analysis at 1.65 A resolution | Vibrio cholerae |
purified recombinant wild-type and mutant G172D enzymes, apoform and Zn2+-bound enzyme, using 400 nl of a precipitant solution containing 0.2 M ammonium acetate, 0.1 M Bis-Tris, pH 5.5, 25% w/v PEG 3350, and 400 nl of 15 mg/ml of protein in crystallization buffer, with or without 1 mM ZnCl2, within 14 days, X-ray diffraction structure determination and analysis at 1.84 A resolution | Haemophilus influenzae |
Protein Variants | Comment | Organism |
---|---|---|
additional information | construction of dimerization domain deletion mutants, that all show no activity | Haemophilus influenzae |
additional information | construction of dimerization domain deletion mutants, that all show no activity | Vibrio cholerae |
T325A | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Haemophilus influenzae |
T325C | site-directed mutagenesis, inactive mutant | Haemophilus influenzae |
T325S | site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme | Haemophilus influenzae |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.8 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant wild-type enzyme | Haemophilus influenzae | |
1.2 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant enzyme | Vibrio cholerae | |
2.1 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant mutant T325A | Haemophilus influenzae | |
3 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant mutant T325S | Haemophilus influenzae |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Zn2+ | required, dinuclear Zn(II)-loaded enzyme, binding structure, overview | Haemophilus influenzae | |
Zn2+ | required, dinuclear Zn(II)-loaded enzyme, binding structure, overview | Vibrio cholerae |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
23200 | - |
catalytic domain, dynamic light scattering | Vibrio cholerae |
28400 | - |
catalytic domain, gel filtration | Haemophilus influenzae |
28700 | - |
catalytic domain, dynamic light scattering | Haemophilus influenzae |
28700 | - |
catalytic domain, gel filtration | Vibrio cholerae |
82600 | - |
wild-type enzyme, gel filtration | Haemophilus influenzae |
83200 | - |
wild-type enzyme, dynamic light scattering | Haemophilus influenzae |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-succinyl-LL-2,6-diaminoheptanedioate + H2O | Haemophilus influenzae | - |
succinate + LL-2,6-diaminoheptanedioate | - |
? | |
N-succinyl-LL-2,6-diaminoheptanedioate + H2O | Vibrio cholerae | - |
succinate + LL-2,6-diaminoheptanedioate | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Haemophilus influenzae | - |
gene dapE | - |
Vibrio cholerae | Q9KQ52 | gene dapE | - |
Purification (Comment) | Organism |
---|---|
recombinant N-terminally His6-tagged enzyme from Escherichia coli strain BL21(DE3)/pMAGIC by nickel affinity chromatography and gel filtration | Vibrio cholerae |
recombinant N-terminally His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3)/pMAGIC by nickel affinity chromatography and gel filtration | Haemophilus influenzae |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
N-succinyl-LL-2,6-diaminoheptanedioate + H2O | - |
Haemophilus influenzae | succinate + LL-2,6-diaminoheptanedioate | - |
? | |
N-succinyl-LL-2,6-diaminoheptanedioate + H2O | - |
Vibrio cholerae | succinate + LL-2,6-diaminoheptanedioate | - |
? |
Subunits | Comment | Organism |
---|---|---|
dimer | the enzyme is composed of catalytic and dimerization domains, the dimerization domain in DapE enzymes is required for catalysis | Haemophilus influenzae |
dimer | the enzyme is composed of catalytic and dimerization domains, the dimerization domain in DapE enzymes is required for catalysis | Vibrio cholerae |
Synonyms | Comment | Organism |
---|---|---|
DapE | - |
Haemophilus influenzae |
DapE | - |
Vibrio cholerae |
N-succinyl-L,L-diaminopimelic acid desuccinylase | - |
Haemophilus influenzae |
N-succinyl-L,L-diaminopimelic acid desuccinylase | - |
Vibrio cholerae |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
25 | - |
assay at | Haemophilus influenzae |
25 | - |
assay at | Vibrio cholerae |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.9 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant mutant T325S | Haemophilus influenzae | |
4 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant mutant T325A | Haemophilus influenzae | |
80 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant enzyme | Vibrio cholerae | |
114 | - |
N-succinyl-LL-2,6-diaminoheptanedioate | pH 7.5, 25°C, recombinant enzyme | Haemophilus influenzae |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
7.5 | - |
assay at | Haemophilus influenzae |
7.5 | - |
assay at | Vibrio cholerae |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family | Haemophilus influenzae |
evolution | the enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family | Vibrio cholerae |
additional information | structural comparisons of wild-type and inactive monomeric DapE enzymes with other M20 peptidases, active site and zinc binding structure, molecular modeling and dynamics simulations, overview. The dimerization domain in DapE enzymes is required for catalysis. Removal of the dimerization domain increases the flexibility of a conserved active site loop that may provide critical interactions with the substrate | Haemophilus influenzae |
additional information | structural comparisons of wild-type and inactive monomeric DapE enzymes with other M20 peptidases, active site and zinc binding structure, molecular modeling and dynamics simulations, overview. The dimerization domain in DapE enzymes is required for catalysis. Removal of the dimerization domain increases the flexibility of a conserved active site loop that may provide critical interactions with the substrate | Vibrio cholerae |