BRENDA - Enzyme Database show

Substrate-mediated stabilization of a tetrameric drug target reveals Achilles heel in anthrax

Voss, J.E.; Scally, S.W.; Taylor, N.L.; Atkinson, S.C.; Griffin, M.D.; Hutton, C.A.; Parker, M.W.; Alderton, M.R.; Gerrard, J.A.; Dobson, R.C.; Dogovski, C.; Perugini, M.A.; J. Biol. Chem. 285, 5188-5195 (2010)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
dapA gene encoding DHDPS amplified and cloned into the pET11a expression vector, expressed in Escherichia coli BL21-DE3
Bacillus anthracis
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
in complex with pyruvate, by sitting- and hanging-drop vapor diffusion method, to 2.15 A resolution, shares the same space group, unit cell parameters, and a similar resolution to the structure of substrate unbound DHDPS. Twelve more hydrogen bond interactions at both interfaces in the crystal structure of pyruvate-bound DHDPS relative to the apo structure
Bacillus anthracis
Engineering
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
L170E/G191E
dimeric mutant of the enzyme, retains only 1.8% of the total catalytic activity of the wild-type tetrameric enzyme
Bacillus anthracis
General Stability
EC Number
General Stability
Organism
4.3.3.7
the secondary and quaternary structure is significantly stabilized in the presence of pyruvate
Bacillus anthracis
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.05
-
L-aspartate 4-semialdehyde
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
0.25
-
L-aspartate 4-semialdehyde
wild-type, at 30°C
Bacillus anthracis
4.3.3.7
1.2
-
pyruvate
wild-type, at 30°C
Bacillus anthracis
4.3.3.7
3.7
-
pyruvate
mutant L170E/G191E, at 30°C
Bacillus anthracis
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.3.3.7
31000
-
2 * 31000, calculated from sequence; 4 * 31000, calculated from sequence
Bacillus anthracis
4.3.3.7
124000
-
wild-type, sequence analysis
Bacillus anthracis
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
4.3.3.7
Bacillus anthracis
Q81WN7
Sterne strain
-
4.3.3.7
Bacillus anthracis Sterne
Q81WN7
Sterne strain
-
Purification (Commentary)
EC Number
Commentary
Organism
4.3.3.7
by sonication, anion-exchange and hydrophobic interaction liquid chromatography
Bacillus anthracis
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
-
704696
Bacillus anthracis
dihydrodipicolinate + 2 H2O
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
4.3.3.7
dimer
2 * 31000, calculated from sequence
Bacillus anthracis
4.3.3.7
More
the tetramer-dimer dissociation constant of the enzyme is 3fold tighter in the presence of pyruvate compared with the apo form
Bacillus anthracis
4.3.3.7
tetramer
4 * 31000, calculated from sequence
Bacillus anthracis
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
4.3.3.7
50
60
thermostability is significantly enhanced in the presence of the substrate pyruvate
Bacillus anthracis
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
4.3.3.7
1.7
-
pyruvate
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
92
-
pyruvate
wild-type, at 30°C
Bacillus anthracis
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
dapA gene encoding DHDPS amplified and cloned into the pET11a expression vector, expressed in Escherichia coli BL21-DE3
Bacillus anthracis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
in complex with pyruvate, by sitting- and hanging-drop vapor diffusion method, to 2.15 A resolution, shares the same space group, unit cell parameters, and a similar resolution to the structure of substrate unbound DHDPS. Twelve more hydrogen bond interactions at both interfaces in the crystal structure of pyruvate-bound DHDPS relative to the apo structure
Bacillus anthracis
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
4.3.3.7
L170E/G191E
dimeric mutant of the enzyme, retains only 1.8% of the total catalytic activity of the wild-type tetrameric enzyme
Bacillus anthracis
General Stability (protein specific)
EC Number
General Stability
Organism
4.3.3.7
the secondary and quaternary structure is significantly stabilized in the presence of pyruvate
Bacillus anthracis
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.05
-
L-aspartate 4-semialdehyde
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
0.25
-
L-aspartate 4-semialdehyde
wild-type, at 30°C
Bacillus anthracis
4.3.3.7
1.2
-
pyruvate
wild-type, at 30°C
Bacillus anthracis
4.3.3.7
3.7
-
pyruvate
mutant L170E/G191E, at 30°C
Bacillus anthracis
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
4.3.3.7
31000
-
2 * 31000, calculated from sequence; 4 * 31000, calculated from sequence
Bacillus anthracis
4.3.3.7
124000
-
wild-type, sequence analysis
Bacillus anthracis
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
by sonication, anion-exchange and hydrophobic interaction liquid chromatography
Bacillus anthracis
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
-
704696
Bacillus anthracis
dihydrodipicolinate + 2 H2O
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
4.3.3.7
dimer
2 * 31000, calculated from sequence
Bacillus anthracis
4.3.3.7
More
the tetramer-dimer dissociation constant of the enzyme is 3fold tighter in the presence of pyruvate compared with the apo form
Bacillus anthracis
4.3.3.7
tetramer
4 * 31000, calculated from sequence
Bacillus anthracis
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
4.3.3.7
50
60
thermostability is significantly enhanced in the presence of the substrate pyruvate
Bacillus anthracis
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
1.7
-
pyruvate
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
92
-
pyruvate
wild-type, at 30°C
Bacillus anthracis
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.46
-
pyruvate
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
34
-
L-aspartate 4-semialdehyde
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
76.67
-
pyruvate
wild-type, at 30°C
Bacillus anthracis
4.3.3.7
368
-
L-aspartate 4-semialdehyde
wild-type, at 30°C
Bacillus anthracis
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
4.3.3.7
0.46
-
pyruvate
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
34
-
L-aspartate 4-semialdehyde
mutant L170E/G191E, at 30°C
Bacillus anthracis
4.3.3.7
76.67
-
pyruvate
wild-type, at 30°C
Bacillus anthracis
4.3.3.7
368
-
L-aspartate 4-semialdehyde
wild-type, at 30°C
Bacillus anthracis