BRENDA - Enzyme Database show

Mutating the tight-dimer interface of dihydrodipicolinate synthase disrupts the enzyme quaternary structure: toward a monomeric enzyme

Pearce, F.G.; Dobson, R.C.; Weber, A.; Lane, L.A.; McCammon, M.G.; Squire, M.A.; Perugini, M.A.; Jameson, G.B.; Robinson, C.V.; Gerrard, J.A.; Biochemistry 47, 12108-12117 (2008)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
expressed in Escherichia coli, pJG001 plasmid for site-directed mutagenesis, mutant Y107W expressed in dapA-negative strain AT997r-
Escherichia coli
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
mutant Y107W, hanging-drop vapor diffusion method, diffraction to beyond 2.0 A resolution, data collection and refinement statistics, solid-state structure of the mutant enzyme largely unchanged
Escherichia coli
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
Y107F
mutant, site-directed mutagenesis
Escherichia coli
4.3.3.7
Y107W
mutant, site-directed mutagenesis
Escherichia coli
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
(S)-lysine
partial mixed inhibition with respect to (S)-aspartate 4-semialdehyde, partial non-ncompetitive inhibition with respect to pyruvate in wild-type and in Y107W mutant, Y107W mutant still retains over 25% of uninhibited activity, even at high inhibitor concentrations compared to the wild-type enzyme retaining less than 10% of normal activity
Escherichia coli
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.11
-
(S)-aspartate 4-semialdehyde
wild-type
Escherichia coli
4.3.3.7
0.16
-
pyruvate
Y107F mutant
Escherichia coli
4.3.3.7
0.26
-
pyruvate
wild-type
Escherichia coli
4.3.3.7
0.39
-
(S)-aspartate 4-semialdehyde
Y107W mutant
Escherichia coli
4.3.3.7
0.58
-
(S)-aspartate 4-semialdehyde
Y107F mutant
Escherichia coli
4.3.3.7
1.5
-
pyruvate
Y107W mutant
Escherichia coli
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.3.3.7
31290
-
monomer size of mutant Y107W, deduced from sequence
Escherichia coli
4.3.3.7
31310
-
monomer size of wild-type, mass spectrometry
Escherichia coli
4.3.3.7
31320
-
monomer size of mutant Y107W, mass spectrometry
Escherichia coli
4.3.3.7
64000
-
dimer size of mutant Y107W, analytical ultracentrifugation
Escherichia coli
4.3.3.7
123000
-
tetramer size of mutant Y107W, analytical ultracentrifugation
Escherichia coli
4.3.3.7
125300
-
tetramer size of wild-type protein, mass spectrometry, mutant form Y107W reveals a mixture of primarily monomer and tetramer in solution
Escherichia coli
4.3.3.7
125400
-
tetramer size of mutant Y107W, mass spectrometry
Escherichia coli
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Escherichia coli
P0A6L2
-
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
gel filtration
Escherichia coli
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
additional information
-
role of Tyr107 residue in determining the quaternary structure, structural, biophysical, and kinetic studies of the Y107W mutant, catalytic ability and apparent melting temperature reduced by the mutation, tetrameric quaternary structure critical to control specificity, heat stability, and intrinsic activity
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
(S)-aspartate 4-semialdehyde + pyruvate
role of Tyr107 residue, located at the tight-dimer interface between two monomers, participates in a catalytic triad of residues during catalysis
690914
Escherichia coli
dihydrodipicolinate + H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
4.3.3.7
homotetramer
wild-type, mixture of primarily monomer and tetramer in solution determined for mutant form Y107W, hydrodynamic properties of Y107W oligomers calculated by sedimentation velocity analyses
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
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
6.5
-
(S)-aspartate 4-semialdehyde
Y107W mutant
Escherichia coli
4.3.3.7
10.8
-
(S)-aspartate 4-semialdehyde
Y107F mutant
Escherichia coli
4.3.3.7
124
-
(S)-aspartate 4-semialdehyde
wild-type
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
expressed in Escherichia coli, pJG001 plasmid for site-directed mutagenesis, mutant Y107W expressed in dapA-negative strain AT997r-
Escherichia coli
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
mutant Y107W, hanging-drop vapor diffusion method, diffraction to beyond 2.0 A resolution, data collection and refinement statistics, solid-state structure of the mutant enzyme largely unchanged
Escherichia coli
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
Y107F
mutant, site-directed mutagenesis
Escherichia coli
4.3.3.7
Y107W
mutant, site-directed mutagenesis
Escherichia coli
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
4.3.3.7
(S)-lysine
partial mixed inhibition with respect to (S)-aspartate 4-semialdehyde, partial non-ncompetitive inhibition with respect to pyruvate in wild-type and in Y107W mutant, Y107W mutant still retains over 25% of uninhibited activity, even at high inhibitor concentrations compared to the wild-type enzyme retaining less than 10% of normal activity
Escherichia coli
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.11
-
(S)-aspartate 4-semialdehyde
wild-type
Escherichia coli
4.3.3.7
0.16
-
pyruvate
Y107F mutant
Escherichia coli
4.3.3.7
0.26
-
pyruvate
wild-type
Escherichia coli
4.3.3.7
0.39
-
(S)-aspartate 4-semialdehyde
Y107W mutant
Escherichia coli
4.3.3.7
0.58
-
(S)-aspartate 4-semialdehyde
Y107F mutant
Escherichia coli
4.3.3.7
1.5
-
pyruvate
Y107W mutant
Escherichia coli
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.3.3.7
31290
-
monomer size of mutant Y107W, deduced from sequence
Escherichia coli
4.3.3.7
31310
-
monomer size of wild-type, mass spectrometry
Escherichia coli
4.3.3.7
31320
-
monomer size of mutant Y107W, mass spectrometry
Escherichia coli
4.3.3.7
64000
-
dimer size of mutant Y107W, analytical ultracentrifugation
Escherichia coli
4.3.3.7
123000
-
tetramer size of mutant Y107W, analytical ultracentrifugation
Escherichia coli
4.3.3.7
125300
-
tetramer size of wild-type protein, mass spectrometry, mutant form Y107W reveals a mixture of primarily monomer and tetramer in solution
Escherichia coli
4.3.3.7
125400
-
tetramer size of mutant Y107W, mass spectrometry
Escherichia coli
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
gel filtration
Escherichia coli
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
additional information
-
role of Tyr107 residue in determining the quaternary structure, structural, biophysical, and kinetic studies of the Y107W mutant, catalytic ability and apparent melting temperature reduced by the mutation, tetrameric quaternary structure critical to control specificity, heat stability, and intrinsic activity
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
(S)-aspartate 4-semialdehyde + pyruvate
role of Tyr107 residue, located at the tight-dimer interface between two monomers, participates in a catalytic triad of residues during catalysis
690914
Escherichia coli
dihydrodipicolinate + H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.3.3.7
homotetramer
wild-type, mixture of primarily monomer and tetramer in solution determined for mutant form Y107W, hydrodynamic properties of Y107W oligomers calculated by sedimentation velocity analyses
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
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
6.5
-
(S)-aspartate 4-semialdehyde
Y107W mutant
Escherichia coli
4.3.3.7
10.8
-
(S)-aspartate 4-semialdehyde
Y107F mutant
Escherichia coli
4.3.3.7
124
-
(S)-aspartate 4-semialdehyde
wild-type
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