Information on EC 5.1.1.7 - diaminopimelate epimerase

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The expected taxonomic range for this enzyme is: Bacteria, Eukaryota

EC NUMBER
COMMENTARY hide
5.1.1.7
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RECOMMENDED NAME
GeneOntology No.
diaminopimelate epimerase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
LL-2,6-diaminoheptanedioate = meso-diaminoheptanedioate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
epimerization
racemization
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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Biosynthesis of secondary metabolites
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L-lysine biosynthesis I
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L-lysine biosynthesis II
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L-lysine biosynthesis VI
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Lysine biosynthesis
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lysine metabolism
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
LL-2,6-diaminoheptanedioate 2-epimerase
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CAS REGISTRY NUMBER
COMMENTARY hide
9024-22-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
gene dapF
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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-
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Manually annotated by BRENDA team
natural excretor of lysine, lysine-overproducing homoserine auxotroph strain and its auxotrophic and multi-analogue-resistant high-yielding mutant AEC NV 20r50
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Manually annotated by BRENDA team
Chlamydomonas sp.
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-
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
gene dapF1; gene dapF1
UniProt
Manually annotated by BRENDA team
gene dapF1; gene dapF1
UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
no activity in Bacillus globisporus
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Manually annotated by BRENDA team
no activity in Bacillus pasteurii
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Manually annotated by BRENDA team
no activity in Bacillus sphaericus
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Manually annotated by BRENDA team
Mixed rumen bacteria
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
additional information
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dimerization of bacterial diaminopimelate epimerase is essential for catalysis, the enzyme exists in an open, active conformation. The active site of the enzyme resides in a cleft between the two domains with each domain contributing one of the cysteine residues important for catalysis
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(2S,6S)-2,6-diaminoheptanedioate
meso-diaminoheptanedioate
show the reaction diagram
-
-
-
?
DL-3-fluoro-2,6-diaminopimelic acid
tetrahydrodipicolinic acid + HF
show the reaction diagram
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rapid elimination, enamine product is formed which spontaneously cyclizes to tetrahydrodipicolinic acid
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?
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
LL-3-fluoro-2,6-diaminopimelic acid
tetrahydrodipicolinic acid + HF
show the reaction diagram
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slow elimination of HF
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?
LL-oxa-diaminopimelic acid
meso-oxa-diaminopimelic acid
show the reaction diagram
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-
-
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?
meso-diaminoheptanedioate
LL-2,6-diaminoheptanedioate
show the reaction diagram
additional information
?
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NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(2-(4-amino-4-carboxybutyl) aziridine-2-carboxylic acid)
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AziDAP
1,2-benzisothiazolidine 3-one
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30 nM, complete inhibition
2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate
substrate mimic, irreversible inhibition
2-(4-amino-4-carboxybutyl)aziridine-2-carboxylic acid
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irreversible
2-nitro-5-thiocyanatobenzoate
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30 nM, complete inhibition
3-Chlorodiaminopimelate
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the inhibitor is converted to a tight-binding transition state analog at the active site of this enzyme
3-Fluoro analogs of diaminopimelate
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potent competitive inhibitors
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4-Oxo-1,2,3,4-tetrahydro-pyridine-2,6-dicarboxylic acid
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very poor inhibitor
5,5'-dithiobis(2-nitrobenzoic acid)
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30 nM, complete inhibition
acetone
inactivation by 50% acetone
dilithium (2Z,6S)-2,6-diamino-4-oxohept-2-enedioate
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competitive, IC50: 0.5 mM
DL-aziridino analogues of diaminoheptanedioate
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DL-aziridino diaminopimelic acid, irreversible inhibitor
DL-aziridino-diaminopimelate
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product-like inhibitor, inhibitor mimics the natural substrate, the methylene carbon of the aziridine ring of the 2 diastereomeric inhibitors is covalently bonded to the sulfur atom of Cys73 or Cys217 after the nucleophilic attack of the sulfur on the aziridine ring that irreversibly inhibits the enzyme
hydroxylamine
iodoacetamide
Isoniazid
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Lanthionine isomers
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Lanthionine sulfone
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Lanthionine sulfoxide
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LL-aziridino analogues of diaminoheptanedioate
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LL-aziridino diaminopimelic acid, irreversible inhibitor
LL-aziridino-diaminopimelate
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reactant-like inhibitor, inhibitor mimics the natural substrate, the methylene carbon of the aziridine ring of the 2 diastereomeric inhibitors is covalently bonded to the sulfur atom of Cys73 or Cys217 after the nucleophilic attack of the sulfur on the aziridine ring that irreversibly inhibits the enzyme
Mercurials
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N-Aminodiaminopimelate
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N-Hydroxydiaminopimelate
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Phosphonate analogs of diaminopimelate
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Semicarbazide
additional information
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
thiol compound
additional information
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.16 - 6.7
LL-diaminoheptanedioate
0.07 - 100
meso-diaminoheptanedioate
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
128
LL-2,6-Diaminoheptanedioate
Haemophilus influenzae
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forward reaction
84
LL-diaminoheptanedioate
Escherichia coli
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0.001 - 67
meso-diaminoheptanedioate
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.5
dilithium (2Z,6S)-2,6-diamino-4-oxohept-2-enedioate
Escherichia coli
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competitive, IC50: 0.5 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.205
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crude extract after expression
0.574
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after immobilized metal-affinity chromatography
1.18
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after gel filtration
1.61
purified recombinant His-tagged enzyme, substrate meso-diaminoheptanedioate, pH 6.8, 30°C
4.93
purified recombinant His-tagged enzyme, substrate LL-2,6-diaminoheptanedioate, pH 6.8, 30°C
additional information
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-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6.5 - 8
7.5
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maximum activity of recombinant DapF consisting of silent mutation of the first 10 codons of the open reading frame
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 8.5
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pH 6: about 60% of maximal activity, pH 8.5: about 25% of maximal activity
6.5 - 9
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recombinant DapF consisting of silent mutation of the first 10 codons of the open reading frame
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
45
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the relative rates of epimerization of LL-diaminoheptanedioate at 25°C, 37°C and 45°C are 0.77:1.00:1.15
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
25 - 45
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the relative rates of epimerization of LL-diaminoheptanedioate at 25°C, 37°C and 45°C are 0.77:1.00:1.15
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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Manually annotated by BRENDA team
PDB
SCOP
CATH
ORGANISM
UNIPROT
Acinetobacter baumannii (strain AB307-0294)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30000
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molecular weight of recombinant DapF consisting of silent mutation of the first 10 codons, determined by SDS-PAGE
31030
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predicted, confirmed by SDS-PAGE
45400
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gel filtration
61300
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recombinant enzyme, analytical ultracentrifugation
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
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2 * 31031, recombinant enzyme, mass spectrometry
monomer
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1 * 34000, SDS-PAGE
additional information
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DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state, dimerization of bacterial diaminopimelate epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. The dimer-monomer dissociation constant is 22 nM. The dimerization interfaceof the epimerase occurs between the N-terminal domains of the two monomers
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant enzyme, hanging drop vapour diffusion method, mixing of 0.001 ml of 14 mg/ml protein in 20 mM HEPES, pH 7.0, 100 mM NaCl, and 5 mM DTT, with 0.001 ml of reservoir solution containing 2 M ammonium sulfate, 0.1 M sodium HEPES, pH 7.5, and 4.9-5.1% PEG 400, equilibration against reservoir solution, 25% v/v PEG 400 as a cryoprotectant, method optimization, X-ray diffraction structure determination and analysis at 1.9 A resolution
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in complex with two different isomers of inhibitor 2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate, at 1.95 and 2.3 A resolution. Ligand binding to a cleft between the two domains of the enzyme is accompanied by domain closure with strictly conserved cysteine residues, Cys99 and Cys254, positioned to perform acid/base catalysis via a carbanion stabilization mechanism on the stereogenic alpha-carbon atom of the amino acid. Stereochemical control in catalysis is achieved by means of a highly symmetric catalytic site that can accommodate both the L and D stereogenic centers of DAP at the proximal site, whereas specific interactions at the distal site require only the L configuration
by using the sitting-drop vapour-diffusion method with droplets consisting of 150 nl protein solution and 150 nl reservoir solution, conditions that yield crystals are replicated using the hanging-drop vapourdiffusion method with drops containing 0.002 ml protein solution and 0.002 ml precipitant solution, crystals are obtained in space group P41212 and diffract to 2.0 A resolution, with unit-cell parameters a = b = 89.4, c = 179.6 A
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sitting drop vapour diffusion method, 2.5 A resolution
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wild-type and Y268A mutant enzymes, hanging drop vapor diffusion method, 0.002 ml of 8.0 mg/ml protein in 20 mM Tris, 5 mM DTT, and 5 mM tris(2-carboxyethyl)phosphine, pH 7.8, are mixed with 0.002 ml of precipitant solution containing 0.2 M sodium iodide, 18% w/v PEG 3350, 0.1 M Bis-Tris propane, pH 6.5, 5 mM diaminoheptanedioate, 20°C, cryoprotectant is glycerol 20% v/v, X-ray diffraction structure determination and analysis at 2.0-2.05 A resolution
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co-crystals of the inhibitors LL- and DL-aziridino diaminopimelic acid with diaminopimelate epimerase from Haemophilus influenzae are grown at room temperature by the hanging-drop vapor-diffusion method. Crystals of both complexes are obtained in 2.8 M sodium acetate /0.1 M Hepes (pH 7.0) at a protein concentration of approx. 10 mg/ml in 25 mM Hepes, 5 mM DTT (pH 8.0); crystal structures of diaminopimelate epimerase from Haemophilus influenzae with two different isomers of the irreversible inhibitor and substrate mimic aziridino diaminopimelic acid at 1.35- and 1.70-A resolution are analysed. These structures permit a detailed description of this pyridoxal 5’-phosphate-independent amino acid racemase active site and delineate the electrostatic interactions that control the exquisite substrate selectivity of DAP epimerase. Moreover, the active site shows how deprotonation of the substrates’nonacidic hydrogen at the alpha-carbon by a seemingly weakly basic cysteine residue is facilitated by interactions with two buried alpha-helices
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comparisons of the mutant structures with the structures of the AziDAP inhibitor-bound form reveal that the enzyme adopts an open conformation in the absence of substrates or inhibitors with the two active site cysteines existing as a thiol–thiolate pair. Substrate binding to the C-terminal domain triggers the closure of the N-terminal domain coupled with tight encapsulation of the ligand, stabilization of the conformation of an active site loop containing Cys73 and expulsion of water molecules with concomitant desolvation of the thiolate base; crystallization of C73S and C217S mutant diaminopimelate epimerase enzymes of Haemophilus influenzae are obtained by the hanging-drop vapor diffusion method and submitted to X-ray structure analysis
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hanging-drop vapour-diffusion method, space group C222(1), unit cell parameters a = 98.64 A, b = 113.87 A, c = 64.48 A, 1.75 A resolution
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crystal structure of the ligand-free form refines to a resolution of 2.6 A, 2.5 mM dithiothreitol is present in the crystal drop
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pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5
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2°C, overnight, 0.1 mM 2,3-dimercaptopropan-1-ol, 20% loss of activity
2136
6
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irreversible loss of activity at pH 6 and below. Partially reactivated by thiols
2140
8
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rapid loss of activity when stored in absence of dithiothreitol
2155
8.5
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2°C, overnight, 0.1 mM 2,3-dimercaptopropan-1-ol, 70% loss of activity
2136
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
air oxidation, in absence of reducing thiols, is slower at pH 7 than at pH 8
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denatured on freezing
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ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acetone
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
0-4°C, best storage temperature
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
by immobilized nickel metal-affinity chromatography using 5 ml HisTrap HP columns and by gel filtration as His-tagged enzyme
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by using nickel-chelate affinity chromatography
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partial
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protein is applied to a Ni21-primed chelating sepharose column, and DapF-containing eluate, fractions are dialysed and further purified by anion exchange chromatography on a HiTrap Q Sepharose FF column, representing a yield of 1 mg/L culture
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recombinant His6-tagged enzyme from Escherichia coli strain SoluBL21 by nickel affinity chromatography, tag cleavage by thrombin, another step of nickel affinity chromatography, ultrafiltration, and gel filtration
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recombinant N-terminally His-tagged enzyme from Escherichia coli strain BL21(DE3)pLysS/pET15b by nickel affinity chromatography
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DAP epimerase mutants C73S and C217S from Haemophilus influenzae are cloned and purified
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expressed in Escherichia coli BL21 (DE3)
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expression in Escherichia coli BL21(DE3)
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gene dapF, overexpression of N-terminally His6-tagged enzyme, cloned with a a thrombin cleavage site, in Escherichia coli strain SoluBL21
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gene dapF1, expression of N-terminally His-tagged enzyme in Escherichia coli strain BL21(DE3)pLysS/pET15b
recombinant protein is expressed as His-tegged enzyme in BL21(DE3) Escherichia coli cells
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since previous attempts to express the diaminopimelate epimerase gene dapF of Mycobacterium tuberculosis in Escherichia coli results in undetectable enzyme yields a recombinant DapF protein is expressed in Escherichia coli consisting of silent mutation of the first 10 codons of the open reading frame in an attempt to reduce the formation of secondary structures that occur near the 5' end of the mRNA and inhibit translation. This significantly increases the yield of the enzyme
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Y268A
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site-directed mutagenesis, the monomeric mutant is catalytically inactive
C217A
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mutant enzyme is inactive as epimerase, catalyzes elimination of HF via abstraction of the C-2 hydrogen from L,L-3-fluoro-2,6-diaminopimelate, incapable of catalyzing HF elimination from D,L-3-fluoro-2,6-diaminopimelate
C217S
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catalyzes epimerization of L,L-diaminopimelate at 2% of the activity of the wild-type enzyme,catalyzes HF elimination from L,L-3-fluoro-2,6-diaminopimelate and D,L-3-fluoro-2,6-diaminopimelate
C73A
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mutant enzyme is inactive as epimerase, catalyzes elimination of HF via abstraction of the C-2 hydrogen. Mutant enzyme is able to rapidly catalyze elimination of the D,L-3-fluoro-2,6-diaminopimelate and is unable to catalyze elimination with the L,L-3-fluoro-2,6-diaminopimelate
C73S
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epimerization of L,L-diaminopimelate at 3% of the activity of the wild-type enzyme, catalyzes HF elimination from L,L-3-fluoro-2,6-diaminopimelate and D,L-3-fluoro-2,6-diaminopimelate
C73S/C217S
C226A
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complete loss of activity
C226S
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severely compromised catalytic efficiency
C87A
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complete loss of activity
C87S
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severely compromised catalytic efficiency despite decrease in Km value
C226A
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complete loss of activity
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C226S
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severely compromised catalytic efficiency
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C87A
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complete loss of activity
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C87S
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severely compromised catalytic efficiency despite decrease in Km value
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additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
drug development
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