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Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine

Muscroft-Taylor, A.C.; Catchpole, R.J.; Dobson, R.C.; Pearce, F.G.; Perugini, M.A.; Gerrard, J.A.; Arch. Biochem. Biophys. 503, 202-206 (2010)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Star (DE3)
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
L197D/Y107W
site-directed mutagenesis, the mutant forms a monomer that is catalytically active, but with reduced catalytic efficiency, displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increase by an order of magnitude. L197D/Y107W is expressed as a folded monomer
Escherichia coli
4.3.3.7
additional information
disruption of quaternary structure of DHDPS generates a functional monomer that is no longer inhibited by lysine, overview
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
L-lysine
inhibition of the tetrameric wild-type enzyme, but not of the disrupted minimeric mutant enzyme. Allosteric binding by two molecules of (S)-lysine at the DHDPS tight-dimer interface cleft has been observed to operate via a cooperative mechanism and to result in incomplete partial mixed inhibition, inhibition kinetics, overview
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
additional information
-
additional information
kinetic study: the wild-type enzyme shows a ping pong mechanism, while the monomeric mutant L197D/Y107W shows ternary-complex mechanism
Escherichia coli
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
L-aspartate 4-semialdehyde + pyruvate
Escherichia coli
-
(S)-2,3-dihydropyridine-2,6-dicarboxylate + 2 H2O
-
-
?
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Escherichia coli
-
gene dapA
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 Star (DE3) by nickel affinity chromatography and gel filtration
Escherichia coli
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
4.3.3.7
L-aspartate 4-semialdehyde + pyruvate
-
713953
Escherichia coli
(S)-2,3-dihydropyridine-2,6-dicarboxylate + 2 H2O
-
-
-
?
4.3.3.7
additional information
the quaternary structure plays a significant role in substrate specificity, overview
713953
Escherichia coli
?
-
-
-
-
Subunits
EC Number
Subunits
Commentary
Organism
4.3.3.7
More
disruption of quaternary structure of DHDPS generates a functional monomer that is no longer inhibited by lysine, overview
Escherichia coli
4.3.3.7
tetramer
-
Escherichia coli
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
4.3.3.7
30
-
assay at
Escherichia coli
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
4.3.3.7
20
95
purified recombinnat wild-type enzyme
Escherichia coli
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
9.8
-
L-aspartate 4-semialdehyde
pH 8.0, 30°C, recombinant wild-type enzyme
Escherichia coli
4.3.3.7
9.8
-
pyruvate
pH 8.0, 30°C, recombinant wild-type enzyme
Escherichia coli
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.3.3.7
8
-
assay at
Escherichia coli
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
gene dapA, overexpression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21 Star (DE3)
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
L197D/Y107W
site-directed mutagenesis, the mutant forms a monomer that is catalytically active, but with reduced catalytic efficiency, displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increase by an order of magnitude. L197D/Y107W is expressed as a folded monomer
Escherichia coli
4.3.3.7
additional information
disruption of quaternary structure of DHDPS generates a functional monomer that is no longer inhibited by lysine, overview
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
L-lysine
inhibition of the tetrameric wild-type enzyme, but not of the disrupted minimeric mutant enzyme. Allosteric binding by two molecules of (S)-lysine at the DHDPS tight-dimer interface cleft has been observed to operate via a cooperative mechanism and to result in incomplete partial mixed inhibition, inhibition kinetics, overview
Escherichia coli
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
kinetic study: the wild-type enzyme shows a ping pong mechanism, while the monomeric mutant L197D/Y107W shows ternary-complex mechanism
Escherichia coli
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
L-aspartate 4-semialdehyde + pyruvate
Escherichia coli
-
(S)-2,3-dihydropyridine-2,6-dicarboxylate + 2 H2O
-
-
?
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21 Star (DE3) by nickel affinity chromatography and gel filtration
Escherichia coli
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
L-aspartate 4-semialdehyde + pyruvate
-
713953
Escherichia coli
(S)-2,3-dihydropyridine-2,6-dicarboxylate + 2 H2O
-
-
-
?
4.3.3.7
additional information
the quaternary structure plays a significant role in substrate specificity, overview
713953
Escherichia coli
?
-
-
-
-
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.3.3.7
More
disruption of quaternary structure of DHDPS generates a functional monomer that is no longer inhibited by lysine, overview
Escherichia coli
4.3.3.7
tetramer
-
Escherichia coli
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
4.3.3.7
30
-
assay at
Escherichia coli
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
4.3.3.7
20
95
purified recombinnat wild-type enzyme
Escherichia coli
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
9.8
-
L-aspartate 4-semialdehyde
pH 8.0, 30°C, recombinant wild-type enzyme
Escherichia coli
4.3.3.7
9.8
-
pyruvate
pH 8.0, 30°C, recombinant wild-type enzyme
Escherichia coli
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
4.3.3.7
8
-
assay at
Escherichia coli
General Information
EC Number
General Information
Commentary
Organism
4.3.3.7
additional information
monomeric species exhibit an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity
Escherichia coli
4.3.3.7
physiological function
DHDPS catalyses a branch point reaction the condensation of pyruvate and (S)-aspartate beta-semialdehyde to form an unstable product, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid, which is ultimately advanced to the final metabolites (S)-lysine and meso-diaminopimelate
Escherichia coli
General Information (protein specific)
EC Number
General Information
Commentary
Organism
4.3.3.7
additional information
monomeric species exhibit an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity
Escherichia coli
4.3.3.7
physiological function
DHDPS catalyses a branch point reaction the condensation of pyruvate and (S)-aspartate beta-semialdehyde to form an unstable product, (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid, which is ultimately advanced to the final metabolites (S)-lysine and meso-diaminopimelate
Escherichia coli