Literature summary for 2.3.1.117 extracted from

Schnell, R.; Oehlmann, W.; Sandalova, T.; Braun, Y.; Huck, C.; Maringer, M.; Singh, M.; Schneider, G.
Tetrahydrodipicolinate N-succinyltransferase and dihydrodipicolinate synthase from Pseudomonas aeruginosa: structure analysis and gene deletion (2012), PLoS ONE, 7, e31133.

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Application

Application	Comment	Organism
additional information	DapA is not an optimal target for drug development against Pseudomonas aeruginosa	Pseudomonas aeruginosa

Cloned(Commentary)

Cloned (Comment)	Organism
gene dapD, phylogenetic tree, expression of His-tagged DapD in Escherichia coli strain BL21(DE3)	Pseudomonas aeruginosa

Crystallization (Commentary)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Inhibitors

Inhibitors	Comment	Organism	Structure
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]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	reaction kinetics for DapD, overview	Pseudomonas aeruginosa
7	-	(S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate	pH 7.5, 22°C	Pseudomonas aeruginosa

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	-	Pseudomonas aeruginosa

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
110000	-	recombinant DapD, gel filtration	Pseudomonas aeruginosa

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
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	-	?

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas aeruginosa	-	gene dapD	-

Purification (Commentary)

Purification (Comment)	Organism
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

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
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	Pseudomonas aeruginosa	?	-	?
succinyl-CoA + (S)-2,3,4,5-tetrahydropyridine-2,6-dicarboxylate + H2O	the enzyme is absolutely specific for the L-2-aminopimelate enantiomer	Pseudomonas aeruginosa	CoA + N-succinyl-L-2-amino-6-oxoheptanedioate	-	?
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	Pseudomonas aeruginosa	CoA + N-succinyl-L-2-amino-6-oxoheptanedioate	-	?

Subunits

Subunits	Comment	Organism
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

Synonyms

Synonyms	Comment	Organism
DapD	-	Pseudomonas aeruginosa
PA3666	-	Pseudomonas aeruginosa
tetrahydrodipicolinate N-succinyltransferase	-	Pseudomonas aeruginosa

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at	Pseudomonas aeruginosa

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
7.5	-	assay at	Pseudomonas aeruginosa

Cofactor

Cofactor	Comment	Organism	Structure
succinyl-CoA	-	Pseudomonas aeruginosa

IC50 Value

IC50 Value	IC50 Value Maximum	Comment	Organism	Inhibitor	Structure
20	-	pH 7.5, 22°C	Pseudomonas aeruginosa	D-2-aminopimelate

General Information

General Information	Comment	Organism
evolution	the DAP biosynthesis pathway is present in most Gram-negative bacteria and mycobacteria	Pseudomonas aeruginosa
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