BRENDA - Enzyme Database show

Expression, purification, crystallization and preliminary X-ray diffraction analysis of dihydrodipicolinate synthase from Bacillus anthracis in the presence of pyruvate

Voss, J.E.; Scally, S.W.; Taylor, N.L.; Dogovski, C.; Alderton, M.R.; Hutton, C.A.; Gerrard, J.A.; Parker, M.W.; Dobson, R.C.; Perugini, M.A.; Acta Crystallogr. Sect. F 65, 188-191 (2009)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
4.3.3.7
PCR product ligated into pCRBluntII-TOPO and transformed into Escherichia coli One Shot TOP10. The dapA insert then amplified, the product, which now contains NdeI and BamHI restriction-endonuclease sites, ligated into pCR-BluntII-TOPO and transformed into Escherichia coli One Shot TOP10, which enables efficient ligation into an expression vector. DapA and linearized pET-11a ligated with T4 DNA ligase and transformed into Escherichia coli BL21(DE3)
Bacillus anthracis
Crystallization (Commentary)
EC Number
Crystallization
Organism
4.3.3.7
in the presence of pyruvate, the hanging-drop vapour-diffusion method, at a resolution of 2.15 A. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.5, b = 124.6, c = 131.0 A, beta = 90.0
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 gel filtration, recombinant enzyme in the absence of the substrate pyruvate (with a yield of 36.3%) and presence of the substrate pyruvate (with a yield of 98%)
Bacillus anthracis
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
-
DHDPS purified in the presence of pyruvate yields a greater amount of recombinant enzyme with 22fold greater specific activity compared with the enzyme purified in the absence of substrate
Bacillus anthracis
4.3.3.7
1.5
-
purified enzyme, recombinant enzyme in the absence of the substrate pyruvate
Bacillus anthracis
4.3.3.7
2
-
crude extract, recombinant enzyme in the absence of the substrate pyruvate
Bacillus anthracis
4.3.3.7
12
-
crude extract, recombinant enzyme in the presence of the substrate pyruvate
Bacillus anthracis
4.3.3.7
33
-
purified enzyme, recombinant enzyme in the presence of the substrate pyruvate
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
-
701513
Bacillus anthracis
dihydrodipicolinate + 2 H2O
-
-
-
?
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
PCR product ligated into pCRBluntII-TOPO and transformed into Escherichia coli One Shot TOP10. The dapA insert then amplified, the product, which now contains NdeI and BamHI restriction-endonuclease sites, ligated into pCR-BluntII-TOPO and transformed into Escherichia coli One Shot TOP10, which enables efficient ligation into an expression vector. DapA and linearized pET-11a ligated with T4 DNA ligase and transformed into Escherichia coli BL21(DE3)
Bacillus anthracis
Crystallization (Commentary) (protein specific)
EC Number
Crystallization
Organism
4.3.3.7
in the presence of pyruvate, the hanging-drop vapour-diffusion method, at a resolution of 2.15 A. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 84.5, b = 124.6, c = 131.0 A, beta = 90.0
Bacillus anthracis
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
4.3.3.7
by gel filtration, recombinant enzyme in the absence of the substrate pyruvate (with a yield of 36.3%) and presence of the substrate pyruvate (with a yield of 98%)
Bacillus anthracis
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
-
DHDPS purified in the presence of pyruvate yields a greater amount of recombinant enzyme with 22fold greater specific activity compared with the enzyme purified in the absence of substrate
Bacillus anthracis
4.3.3.7
1.5
-
purified enzyme, recombinant enzyme in the absence of the substrate pyruvate
Bacillus anthracis
4.3.3.7
2
-
crude extract, recombinant enzyme in the absence of the substrate pyruvate
Bacillus anthracis
4.3.3.7
12
-
crude extract, recombinant enzyme in the presence of the substrate pyruvate
Bacillus anthracis
4.3.3.7
33
-
purified enzyme, recombinant enzyme in the presence of the substrate pyruvate
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
-
701513
Bacillus anthracis
dihydrodipicolinate + 2 H2O
-
-
-
?