BRENDA - Enzyme Database show

Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion

Schnell, R.; Oehlmann, W.; Sandalova, T.; Braun, Y.; Huck, C.; Maringer, M.; Singh, M.; Schneider, G.; PLoS ONE 7, e31133 (2012)

Data extracted from this reference:

Application
EC Number
Application
Commentary
Organism
2.3.1.117
additional information
DapA is not an optimal target for drug development against Pseudomonas aeruginosa
Pseudomonas aeruginosa
4.3.3.7
additional information
the enzyme is not an optimal target for drug development against Pseudomonas aeruginosa
Pseudomonas aeruginosa
Cloned(Commentary)
EC Number
Commentary
Organism
2.3.1.117
gene dapD, phylogenetic tree, expression of His-tagged DapD in Escherichia coli strain BL21(DE3)
Pseudomonas aeruginosa
4.3.3.7
gene dapA, expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
Pseudomonas aeruginosa
Crystallization (Commentary)
EC Number
Crystallization
Organism
2.3.1.117
purified recombinant His6-tagged DapD with bound L-2-aminopimelate and D-2-aminopimelate or in complex with CoA/succinate, hanging drop vapour diffusion method, mixing of 0.002 ml of 26 mg/ml protein in 25 mM Tris-HCl pH 8.0, and 150 mM NaCl, with or without 10-15 mM CoA, with 0.002 ml of reservoir solution containing 19-20% of PEG3350, 0.3-0.4 M succinate, pH 6.2, and equilibration against reservoir solution for 1-2 days, incubation of the enzyme with formyl-CoA leads to better crystals, soaking of apoenzyme crystals in solution containing L-2-aminopimelate and D-2-aminopimelate, the CoA-complex also contains a succinatemolecule bound next to the acceptor arm of the CoA in the active site cleft, X-ray diffraction structure determination and analysis at 1.8-2.95 A resolution, molecular replacement
Pseudomonas aeruginosa
4.3.3.7
purified recombinant His6-tagged enzyme, hanging drop vapour diffusion method, mixing of 0.002 m of 12.5 mg/ml protein solution with 0.002 ml of reservoir solution containing 18% of PEG6000, 0.2 M MgCl2, and 0.1 M TRIS-HCl, pH 7.6, X-ray diffraction structure determination and analysis at 1.6 A resolution, molecular replacement
Pseudomonas aeruginosa
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
additional information
mutant construction by gene replacement of gene dapA (PA1010) via the sacB-based strategy
Pseudomonas aeruginosa
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
2.3.1.117
D-2-aminopimelate
very weak competitive inhibition, L-2-aminopimelate and D-2-aminopimelate bind at the same site of the enzyme. Binding interaction analysis of the ligands in the enzyme active site suggests a misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition
Pseudomonas aeruginosa
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.3.1.117
additional information
-
additional information
reaction kinetics for DapD, overview
Pseudomonas aeruginosa
2.3.1.117
7
-
(S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate
pH 7.5, 22°C
Pseudomonas aeruginosa
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
2.3.1.117
Mg2+
-
Pseudomonas aeruginosa
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.3.1.117
110000
-
recombinant DapD, gel filtration
Pseudomonas aeruginosa
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.3.1.117
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O
Pseudomonas aeruginosa
the enzyme is absolutely specific for the L-2-aminopimelate enantiomer
CoA + N-succinyl-L-2-amino-6-oxoheptanedioate
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Pseudomonas aeruginosa
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
2.3.1.117
Pseudomonas aeruginosa
-
gene dapD
-
4.3.3.7
Pseudomonas aeruginosa
Q9I4W3
gene dapA or PA1010
-
Purification (Commentary)
EC Number
Commentary
Organism
2.3.1.117
recombinant His-tagged DapD from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, removal of te the N-terminal His6-tag by thrombin cleavage is not successful
Pseudomonas aeruginosa
4.3.3.7
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, the proteolytic cleavage by TEV protease does not remove the N-terminal His6-tag efficiently
Pseudomonas aeruginosa
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.3.1.117
additional information
no activity ith L-lysine, adipic acid, alpha-amino-adipic acid, L-epsilon-acetyl-lysine, L-glutamate, L-glutamine, L-norleucine, substrate specificity for DapD, overview. Binding of CoA to PaDapD does not induce any large conformational changes, ternary complex structure of DapD with bound CoA and succinate, overview
720852
Pseudomonas aeruginosa
?
-
-
-
-
2.3.1.117
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O
the enzyme is absolutely specific for the L-2-aminopimelate enantiomer
720852
Pseudomonas aeruginosa
CoA + N-succinyl-L-2-amino-6-oxoheptanedioate
-
-
-
?
2.3.1.117
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O
the enzyme is absolutely specific for the L-2-aminopimelate enantiomer, L-2-aminopimelate and weak inhibitor D-2-aminopimelate bind at the same site of the enzyme. Binding interaction analysis of the ligands in the enzyme active site suggests a misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition
720852
Pseudomonas aeruginosa
CoA + N-succinyl-L-2-amino-6-oxoheptanedioate
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
720852
Pseudomonas aeruginosa
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
2.3.1.117
trimer
the subunit of PaDapD consists of three domains, the N-terminal globular domain, a central domain, and a C-terminal domain, overview
Pseudomonas aeruginosa
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.3.1.117
22
-
assay at
Pseudomonas aeruginosa
4.3.3.7
22
-
assay at
Pseudomonas aeruginosa
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.3.1.117
7.5
-
assay at
Pseudomonas aeruginosa
4.3.3.7
8
-
assay at
Pseudomonas aeruginosa
Cofactor
EC Number
Cofactor
Commentary
Organism
Structure
2.3.1.117
succinyl-CoA
-
Pseudomonas aeruginosa
IC50 Value
EC Number
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
2.3.1.117
20
-
pH 7.5, 22°C
Pseudomonas aeruginosa
D-2-aminopimelate
Application (protein specific)
EC Number
Application
Commentary
Organism
2.3.1.117
additional information
DapA is not an optimal target for drug development against Pseudomonas aeruginosa
Pseudomonas aeruginosa
4.3.3.7
additional information
the enzyme is not an optimal target for drug development against Pseudomonas aeruginosa
Pseudomonas aeruginosa
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
2.3.1.117
gene dapD, phylogenetic tree, expression of His-tagged DapD in Escherichia coli strain BL21(DE3)
Pseudomonas aeruginosa
4.3.3.7
gene dapA, expression of N-terminally His6-tagged enzyme in Escherichia coli strain BL21(DE3)
Pseudomonas aeruginosa
Cofactor (protein specific)
EC Number
Cofactor
Commentary
Organism
Structure
2.3.1.117
succinyl-CoA
-
Pseudomonas aeruginosa
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
2.3.1.117
purified recombinant His6-tagged DapD with bound L-2-aminopimelate and D-2-aminopimelate or in complex with CoA/succinate, hanging drop vapour diffusion method, mixing of 0.002 ml of 26 mg/ml protein in 25 mM Tris-HCl pH 8.0, and 150 mM NaCl, with or without 10-15 mM CoA, with 0.002 ml of reservoir solution containing 19-20% of PEG3350, 0.3-0.4 M succinate, pH 6.2, and equilibration against reservoir solution for 1-2 days, incubation of the enzyme with formyl-CoA leads to better crystals, soaking of apoenzyme crystals in solution containing L-2-aminopimelate and D-2-aminopimelate, the CoA-complex also contains a succinatemolecule bound next to the acceptor arm of the CoA in the active site cleft, X-ray diffraction structure determination and analysis at 1.8-2.95 A resolution, molecular replacement
Pseudomonas aeruginosa
4.3.3.7
purified recombinant His6-tagged enzyme, hanging drop vapour diffusion method, mixing of 0.002 m of 12.5 mg/ml protein solution with 0.002 ml of reservoir solution containing 18% of PEG6000, 0.2 M MgCl2, and 0.1 M TRIS-HCl, pH 7.6, X-ray diffraction structure determination and analysis at 1.6 A resolution, molecular replacement
Pseudomonas aeruginosa
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
additional information
mutant construction by gene replacement of gene dapA (PA1010) via the sacB-based strategy
Pseudomonas aeruginosa
IC50 Value (protein specific)
EC Number
IC50 Value
IC50 Value Maximum
Commentary
Organism
Inhibitor
Structure
2.3.1.117
20
-
pH 7.5, 22°C
Pseudomonas aeruginosa
D-2-aminopimelate
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
2.3.1.117
D-2-aminopimelate
very weak competitive inhibition, L-2-aminopimelate and D-2-aminopimelate bind at the same site of the enzyme. Binding interaction analysis of the ligands in the enzyme active site suggests a misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition
Pseudomonas aeruginosa
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
2.3.1.117
additional information
-
additional information
reaction kinetics for DapD, overview
Pseudomonas aeruginosa
2.3.1.117
7
-
(S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate
pH 7.5, 22°C
Pseudomonas aeruginosa
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
2.3.1.117
Mg2+
-
Pseudomonas aeruginosa
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
2.3.1.117
110000
-
recombinant DapD, gel filtration
Pseudomonas aeruginosa
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
2.3.1.117
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O
Pseudomonas aeruginosa
the enzyme is absolutely specific for the L-2-aminopimelate enantiomer
CoA + N-succinyl-L-2-amino-6-oxoheptanedioate
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Pseudomonas aeruginosa
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
2.3.1.117
recombinant His-tagged DapD from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, removal of te the N-terminal His6-tag by thrombin cleavage is not successful
Pseudomonas aeruginosa
4.3.3.7
recombinant His6-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, the proteolytic cleavage by TEV protease does not remove the N-terminal His6-tag efficiently
Pseudomonas aeruginosa
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
2.3.1.117
additional information
no activity ith L-lysine, adipic acid, alpha-amino-adipic acid, L-epsilon-acetyl-lysine, L-glutamate, L-glutamine, L-norleucine, substrate specificity for DapD, overview. Binding of CoA to PaDapD does not induce any large conformational changes, ternary complex structure of DapD with bound CoA and succinate, overview
720852
Pseudomonas aeruginosa
?
-
-
-
-
2.3.1.117
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O
the enzyme is absolutely specific for the L-2-aminopimelate enantiomer
720852
Pseudomonas aeruginosa
CoA + N-succinyl-L-2-amino-6-oxoheptanedioate
-
-
-
?
2.3.1.117
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O
the enzyme is absolutely specific for the L-2-aminopimelate enantiomer, L-2-aminopimelate and weak inhibitor D-2-aminopimelate bind at the same site of the enzyme. Binding interaction analysis of the ligands in the enzyme active site suggests a misalignment of the amino group of D-2-aminopimelate for nucleophilic attack on the succinate moiety of the co-substrate succinyl-CoA as the structural basis of specificity and inhibition
720852
Pseudomonas aeruginosa
CoA + N-succinyl-L-2-amino-6-oxoheptanedioate
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
720852
Pseudomonas aeruginosa
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
2.3.1.117
trimer
the subunit of PaDapD consists of three domains, the N-terminal globular domain, a central domain, and a C-terminal domain, overview
Pseudomonas aeruginosa
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
2.3.1.117
22
-
assay at
Pseudomonas aeruginosa
4.3.3.7
22
-
assay at
Pseudomonas aeruginosa
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
2.3.1.117
7.5
-
assay at
Pseudomonas aeruginosa
4.3.3.7
8
-
assay at
Pseudomonas aeruginosa
General Information
EC Number
General Information
Commentary
Organism
2.3.1.117
evolution
the DAP biosynthesis pathway is present in most Gram-negative bacteria and mycobacteria
Pseudomonas aeruginosa
2.3.1.117
metabolism
tetrahydrodipicolinate N-succinyltransferase catalyses the transfer of the succinyl moiety of succinyl-CoA to the alpha-amino group of tetrahydrodipicolinate, the first committed step in the succinylase branch of the DAP biosynthesis pathway, diaminopimelic acid pathway of lysine biosynthesis, overview
Pseudomonas aeruginosa
4.3.3.7
malfunction
enzyme mutants with deleted dapA gene are viable and able to grow in a mouse lung infection model
Pseudomonas aeruginosa
4.3.3.7
metabolism
the enzyme catalyzes the first step in the diaminopimelic acid pathway of lysine biosynthesis
Pseudomonas aeruginosa
4.3.3.7
additional information
structure-based sequence alignments, based on the DapA crystal structure, reveal the presence of two homologues, PA0223 and PA4188, in Pseudomonas aeruginosa that can substitute for DapA in the PAO1DELTAdapA mutant. In vitro experiments using recombinant PA0223 protein do not detect any DapA activity
Pseudomonas aeruginosa
General Information (protein specific)
EC Number
General Information
Commentary
Organism
2.3.1.117
evolution
the DAP biosynthesis pathway is present in most Gram-negative bacteria and mycobacteria
Pseudomonas aeruginosa
2.3.1.117
metabolism
tetrahydrodipicolinate N-succinyltransferase catalyses the transfer of the succinyl moiety of succinyl-CoA to the alpha-amino group of tetrahydrodipicolinate, the first committed step in the succinylase branch of the DAP biosynthesis pathway, diaminopimelic acid pathway of lysine biosynthesis, overview
Pseudomonas aeruginosa
4.3.3.7
malfunction
enzyme mutants with deleted dapA gene are viable and able to grow in a mouse lung infection model
Pseudomonas aeruginosa
4.3.3.7
metabolism
the enzyme catalyzes the first step in the diaminopimelic acid pathway of lysine biosynthesis
Pseudomonas aeruginosa
4.3.3.7
additional information
structure-based sequence alignments, based on the DapA crystal structure, reveal the presence of two homologues, PA0223 and PA4188, in Pseudomonas aeruginosa that can substitute for DapA in the PAO1DELTAdapA mutant. In vitro experiments using recombinant PA0223 protein do not detect any DapA activity
Pseudomonas aeruginosa