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Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Atkinson, S.C.; Dogovski, C.; Downton, M.T.; Czabotar, P.E.; Dobson, R.C.; Gerrard, J.A.; Wagner, J.; Perugini, M.A.; Plant Mol. Biol. 81, 431-446 (2013)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
Vitis vinifera
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
purified recombinant His-tagged enzyme, hanging drop vapor diffusion method, mixing of 0.002 ml of protein in 20 mM pyruvate and 20 mM lysine, with 0.002 ml of reservoir solution containing 0.1 M MES, pH 6.5, 6% v/v PEG 20000, X-ray diffraction structure determination and analysis at 2.40 A resolution
Vitis vinifera
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
L-lysine
feedback inhibition, binding of lysine to the allosteric cleft of the enzyme, cooperative binding, structural mechanism for allosteric inhibition, overview. With respect to L-aspartate-4-semialdehyde, lysine is a noncompetitive inhibitor
Vitis vinifera
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
additional information
-
additional information
Michaelis-Menten kinetics
Vitis vinifera
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
pyruvate + L-aspartate-4-semialdehyde
Vitis vinifera
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate + H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Vitis vinifera
D7U7T8
gene dapA
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity chromatography
Vitis vinifera
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
the reaction proceeds via a ping-pong kinetic mechanism in which pyruvate binds and forms a Schiff base to an active-site lysine residue (Lys184 in Vitis vinifera enzyme). L-aspartate-4-semialdehyde then reacts with the resultant enamine and following cyclization forms the product (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate
Vitis vinifera
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
additional information
coupled assay with dihydrodipicolinate reductase
730624
Vitis vinifera
?
-
-
-
-
4.3.3.7
pyruvate + L-aspartate-4-semialdehyde
-
730624
Vitis vinifera
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate + H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
4.3.3.7
homotetramer
homotetramer of four identical (beta/alpha)8-barrel monomers, a dimer of tight dimers, with two tight-dimers binding in a head-to-head manner, with the active site situated near the center of the barrel, molecular dynamics simulations
Vitis vinifera
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
4.3.3.7
30
-
assay at
Vitis vinifera
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.3.3.7
8
-
assay at
Vitis vinifera
Ki Value [mM]
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
4.3.3.7
additional information
-
additional information
kinetic and thermodynamics of allosteric inhibitin by lysine, overview
Vitis vinifera
4.3.3.7
0.049
-
L-lysine
versus L-aspartate-4-semialdehyde, pH 8.0, 30°C, recombinant enzyme
Vitis vinifera
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3)
Vitis vinifera
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
purified recombinant His-tagged enzyme, hanging drop vapor diffusion method, mixing of 0.002 ml of protein in 20 mM pyruvate and 20 mM lysine, with 0.002 ml of reservoir solution containing 0.1 M MES, pH 6.5, 6% v/v PEG 20000, X-ray diffraction structure determination and analysis at 2.40 A resolution
Vitis vinifera
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
L-lysine
feedback inhibition, binding of lysine to the allosteric cleft of the enzyme, cooperative binding, structural mechanism for allosteric inhibition, overview. With respect to L-aspartate-4-semialdehyde, lysine is a noncompetitive inhibitor
Vitis vinifera
Ki Value [mM] (protein specific)
EC Number
Ki Value [mM]
Ki Value maximum [mM]
Inhibitor
Commentary
Organism
Structure
4.3.3.7
additional information
-
additional information
kinetic and thermodynamics of allosteric inhibitin by lysine, overview
Vitis vinifera
4.3.3.7
0.049
-
L-lysine
versus L-aspartate-4-semialdehyde, pH 8.0, 30°C, recombinant enzyme
Vitis vinifera
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
additional information
-
additional information
Michaelis-Menten kinetics
Vitis vinifera
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
pyruvate + L-aspartate-4-semialdehyde
Vitis vinifera
-
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate + H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by metal affinity chromatography
Vitis vinifera
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
additional information
coupled assay with dihydrodipicolinate reductase
730624
Vitis vinifera
?
-
-
-
-
4.3.3.7
pyruvate + L-aspartate-4-semialdehyde
-
730624
Vitis vinifera
(2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate + H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.3.3.7
homotetramer
homotetramer of four identical (beta/alpha)8-barrel monomers, a dimer of tight dimers, with two tight-dimers binding in a head-to-head manner, with the active site situated near the center of the barrel, molecular dynamics simulations
Vitis vinifera
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
4.3.3.7
30
-
assay at
Vitis vinifera
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.3.3.7
8
-
assay at
Vitis vinifera
General Information
EC Number
General Information
Commentary
Organism
4.3.3.7
metabolism
lysine biosynthesis in plants is tightly regulated by feedback inhibition of the end product on dihydrodipicolinate synthase, the first enzyme of the lysine-specific branch
Vitis vinifera
General Information (protein specific)
EC Number
General Information
Commentary
Organism
4.3.3.7
metabolism
lysine biosynthesis in plants is tightly regulated by feedback inhibition of the end product on dihydrodipicolinate synthase, the first enzyme of the lysine-specific branch
Vitis vinifera