BRENDA - Enzyme Database show

Cloning, expression, purification and crystallization of dihydrodipicolinate synthase from Agrobacterium tumefaciens

Atkinson, S.C.; Dogovski, C.; Dobson, R.C.; Perugini, M.A.; Acta Crystallogr. Sect. F 68, 1040-1047 (2012)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, sequence comparison with Escherichia coli enzyme, expression of His-tagged enzyme in Escherichia coli strain BL21 (DE3)
Agrobacterium tumefaciens
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
purified recombinant His-tagged enzyme in the unliganded form and in forms with bound substrate and with bound substrate plus allosteric inhibitor lysine, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml protein in 20 mM Tris, pH 8.0, with 0.002 ml of reservoir solution containing 0.1 M Tris, pH 8.5, and 2 M ammonium sulfate, for ligand-bound enzyme from 0.17 M lithium sulfate monohydrate, 0.085 M Tris pH 8.5, 25.5%(w/v) PEG 4000, 15%(v/v) glycerol with addition of 20 mM pyruvate or 20 mM pyruvate and 20 mM lysine, equilibration against 1 ml reservoir solution, 20░C, overnight, X-ray diffraction structure determination and analysis at 1.40-1.55 A resolution
Agrobacterium tumefaciens
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
D-Lysine
allosteric inhibitor
Agrobacterium tumefaciens
Natural Substrates/ Products (Substrates)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Agrobacterium tumefaciens
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Agrobacterium tumefaciens C58 / ATCC 33970
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Agrobacterium tumefaciens
Q8UGL3
gene dapA
-
4.3.3.7
Agrobacterium tumefaciens C58 / ATCC 33970
Q8UGL3
gene dapA
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
recombinant His-tagged enzyme 8fold from Escherichia coli strain BL21 (DE3) by immobilized metal affinity chromatography and gel filtration
Agrobacterium tumefaciens
Reaction
EC Number
Reaction
Commentary
Organism
4.3.3.7
pyruvate + L-aspartate-4-semialdehyde = (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate + H2O
ping-pong mechanism, in which pyruvate binds as a Schiff base to an active-site lysine residue (Lys162 in Agrobacterium tumefaciens)
Agrobacterium tumefaciens
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
4.3.3.7
174
-
purified recombinant His-tagged enzyme, coupled assay, pH and temperature not specified in the publication
Agrobacterium tumefaciens
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
728891
Agrobacterium tumefaciens
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
728891
Agrobacterium tumefaciens C58 / ATCC 33970
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, sequence comparison with Escherichia coli enzyme, expression of His-tagged enzyme in Escherichia coli strain BL21 (DE3)
Agrobacterium tumefaciens
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
purified recombinant His-tagged enzyme in the unliganded form and in forms with bound substrate and with bound substrate plus allosteric inhibitor lysine, hanging drop vapour diffusion method, mixing of 0.002 ml of 10 mg/ml protein in 20 mM Tris, pH 8.0, with 0.002 ml of reservoir solution containing 0.1 M Tris, pH 8.5, and 2 M ammonium sulfate, for ligand-bound enzyme from 0.17 M lithium sulfate monohydrate, 0.085 M Tris pH 8.5, 25.5%(w/v) PEG 4000, 15%(v/v) glycerol with addition of 20 mM pyruvate or 20 mM pyruvate and 20 mM lysine, equilibration against 1 ml reservoir solution, 20░C, overnight, X-ray diffraction structure determination and analysis at 1.40-1.55 A resolution
Agrobacterium tumefaciens
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
D-Lysine
allosteric inhibitor
Agrobacterium tumefaciens
Natural Substrates/ Products (Substrates) (protein specific)
EC Number
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Agrobacterium tumefaciens
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
Agrobacterium tumefaciens C58 / ATCC 33970
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
recombinant His-tagged enzyme 8fold from Escherichia coli strain BL21 (DE3) by immobilized metal affinity chromatography and gel filtration
Agrobacterium tumefaciens
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [Ámol/min/mg]
Specific Activity Maximum [Ámol/min/mg]
Commentary
Organism
4.3.3.7
174
-
purified recombinant His-tagged enzyme, coupled assay, pH and temperature not specified in the publication
Agrobacterium tumefaciens
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
728891
Agrobacterium tumefaciens
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
4.3.3.7
(S)-aspartate-4-semialdehyde + pyruvate
-
728891
Agrobacterium tumefaciens C58 / ATCC 33970
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid + H2O
-
-
-
?
General Information
EC Number
General Information
Commentary
Organism
4.3.3.7
metabolism
the enzyme catalyses the first committed step in the lysine biosynthesis pathway, the condensation of pyruvate and (S)-aspartate semialdehyde to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid
Agrobacterium tumefaciens
General Information (protein specific)
EC Number
General Information
Commentary
Organism
4.3.3.7
metabolism
the enzyme catalyses the first committed step in the lysine biosynthesis pathway, the condensation of pyruvate and (S)-aspartate semialdehyde to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid
Agrobacterium tumefaciens