Information on EC 5.1.1.7 - diaminopimelate epimerase

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

EC NUMBER
COMMENTARY
5.1.1.7
-
RECOMMENDED NAME
GeneOntology No.
diaminopimelate epimerase
REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
LL-2,6-diaminoheptanedioate = meso-diaminoheptanedioate
show the reaction diagram
two-base mechanism for proton translocation. One, but not both, of the proton acceptor sites is a thiol
-
-
-
LL-2,6-diaminoheptanedioate = meso-diaminoheptanedioate
show the reaction diagram
molecular dynamics simulations show that the configuration of the distal carbon C–6 of L,L-DAP is critical for complex formation since both amino and carboxylate groups are involved in H–bonding interactions with the active site residues. Furthermore, the interactions occurring between the functional groups bonded to the C–2 and some residues of the binding cavity immobilize the ligand in a position appropriate for the epimerization reaction, i.e., exactly in the middle of the two catalytic residues Cys73 and Cys217 as confirmed by DFT (density-functional theory) quantum mechanical computation of the Michaelis complex
-
REACTION TYPE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
epimerization
-
-
-
-
epimerization
-
-
racemization
-
-
PATHWAY
KEGG Link
MetaCyc Link
Biosynthesis of secondary metabolites
-
Lysine biosynthesis
-
lysine biosynthesis I
-
lysine biosynthesis II
-
lysine biosynthesis VI
-
Metabolic pathways
-
Microbial metabolism in diverse environments
-
SYSTEMATIC NAME
IUBMB Comments
LL-2,6-diaminoheptanedioate 2-epimerase
-
SYNONYMS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
DAP epimerase
-
-
-
-
DAP epimerase
-
-
DAP epimerase
-
-
DAP-epimerase
-
-
-
-
DAP-epimerase
-
-
diaminopimelate epimerase
-
-
diaminopimelate epimerase
-
-
diaminopimelate epimerase
-
-
Diaminopimelic acid epimerase
-
-
-
-
Diaminopimelic acid epimerase
-
-
Diaminopimelic acid epimerase
-
-
Diaminopimelic epimerase
-
-
-
-
Epimerase, diaminopimelate
-
-
-
-
LL-Diaminopimelate epimerase
-
-
-
-
CAS REGISTRY NUMBER
COMMENTARY
9024-22-0
-
ORGANISM
COMMENTARY
LITERATURE
SEQUENCE CODE
SEQUENCE DB
SOURCE
-
-
-
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
-
-
Manually annotated by BRENDA team
Chlamydomonas sp.
-
-
-
Manually annotated by BRENDA team
no activity in Bacillus globisporus
-
-
-
Manually annotated by BRENDA team
no activity in Bacillus pasteurii
-
-
-
Manually annotated by BRENDA team
no activity in Bacillus sphaericus
-
-
-
Manually annotated by BRENDA team
Mixed rumen bacteria
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
physiological function
-
synthesis of L-lysine
physiological function
-
enzyme is a member of the PLP-independent amino-acid racemases, it catalyzes the penultimate step of lysine biosynthesis in bacteria and plants
physiological function
-
enzyme belongs to the group of isomerases which are capable of inverting the absolute configuration of a carbon atom in substrates containing one (racemases) or more stereocenters
SUBSTRATE
PRODUCT                      
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
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
-
rapid elimination, enamine product is formed which spontaneously cyclizes to tetrahydrodipicolinic acid
-
?
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
P44859
-
-
-
?
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
?
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
?
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
Chlamydomonas sp.
-
-
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
-
r, between 25°C and 45°C at pH 7.0, the equilibrium mixture contains 65% meso-isomer and 35% LL-isomer
-
-
LL-2,6-Diaminoheptanedioate
meso-Diaminoheptanedioate
show the reaction diagram
Bacillus megaterium CII 19
-
-
-
-
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
-
enzyme active in two of three possible pathways for synthesis of L-Lys, acetyltransferase pathway and succinyltransferase pathway. Not active in D-diaminopimelate dehydrogenase variant
-
-
-
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
-
enzyme of Lys biosynthesis
-
-
-
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
-
enzyme of the diaminopimelic acid pathway for biosynthesis of Lys
-
-
-
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
Bacillus megaterium CII 19
-
enzyme of Lys biosynthesis
-
-
-
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
-
stereo-conversion, the product complex (Enzyme/meso-diaminopimelate) is less stable than the reactant complex (Enzyme/LL-diaminopimelate)
-
-
r
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
-
stereo-inversion
-
-
?
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
-
stereo-inversion
the meso-isomer of diaminopimelic acid, a precursor of L-lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan
-
?
LL-3-fluoro-2,6-diaminopimelic acid
tetrahydrodipicolinic acid + HF
show the reaction diagram
-
slow elimination of HF
-
?
LL-oxa-diaminopimelic acid
meso-oxa-diaminopimelic acid
show the reaction diagram
-
-
-
-
?
meso-diaminoheptanedioate
LL-2,6-diaminoheptanedioate
show the reaction diagram
P63897
-
-
-
r
additional information
?
-
-, Q9LFG2
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
-
-
-
NATURAL SUBSTRATES
NATURAL PRODUCTS
REACTION DIAGRAM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
(Substrate)
LITERATURE
(Substrate)
COMMENTARY
(Product)
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
-
enzyme active in two of three possible pathways for synthesis of L-Lys, acetyltransferase pathway and succinyltransferase pathway. Not active in D-diaminopimelate dehydrogenase variant
-
-
-
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
-
enzyme of Lys biosynthesis
-
-
-
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
-
enzyme of the diaminopimelic acid pathway for biosynthesis of Lys
-
-
-
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
-
stereo-conversion, the product complex (Enzyme/meso-diaminopimelate) is less stable than the reactant complex (Enzyme/LL-diaminopimelate)
-
-
r
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
-
stereo-inversion
-
-
?
LL-2,6-diaminoheptanedioate
meso-diaminopimelate
show the reaction diagram
-
stereo-inversion
the meso-isomer of diaminopimelic acid, a precursor of L-lysine, is a key component of the pentapeptide linker in bacterial peptidoglycan
-
?
LL-2,6-Diaminoheptanedioate
?
show the reaction diagram
Bacillus megaterium CII 19
-
enzyme of Lys biosynthesis
-
-
-
INHIBITORS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
(2-(4-amino-4-carboxybutyl) aziridine-2-carboxylic acid)
-
AziDAP
1,2-benzisothiazolidine 3-one
-
30 nM, complete inhibition
2-(4-amino-4-carboxybutyl)-aziridine-2-carboxylate
-, Q9LFG2
substrate mimic, irreversible inhibition
2-(4-amino-4-carboxybutyl)aziridine-2-carboxylic acid
-
irreversible
2-nitro-5-thiocyanatobenzoate
-
30 nM, complete inhibition
3-Chlorodiaminopimelate
-
the inhibitor is converted to a tight-binding transition state analog at the active site of this enzyme
3-Fluoro analogs of diaminopimelate
-
potent competitive inhibitors
-
4-Oxo-1,2,3,4-tetrahydro-pyridine-2,6-dicarboxylic acid
-
very poor inhibitor
5,5'-dithiobis(2-nitrobenzoic acid)
-
30 nM, complete inhibition
dilithium (2Z,6S)-2,6-diamino-4-oxohept-2-enedioate
-
competitive, IC50: 0.5 mM
DL-aziridino analogues of diaminoheptanedioate
-
DL-aziridino diaminopimelic acid, irreversible inhibitor
DL-aziridino-diaminopimelate
-
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
-
no inhibition
hydroxylamine
-
inhibition; no inhibition
iodoacetamide
-
half-life for inactivation with 0.25 mM iodoacetamide is 9.6 min
iodoacetamide
-
-
Lanthionine isomers
-
-
-
Lanthionine sulfone
-
-
Lanthionine sulfoxide
-
-
LL-aziridino analogues of diaminoheptanedioate
-
LL-aziridino diaminopimelic acid, irreversible inhibitor
LL-aziridino-diaminopimelate
-
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
N-Aminodiaminopimelate
-
-
N-Hydroxydiaminopimelate
-
-
Phosphonate analogs of diaminopimelate
-
-
-
Semicarbazide
-
no inhibition
Semicarbazide
-
-
Mercurials
-
-
-
additional information
-
no inhibition by deoxypyridioxine
-
additional information
-
-
-
additional information
-
none of the following compounds shows significant inhibition: (2S)-2-amino-3-(4-carboxyimidazol-1-yl)propanoic acid, (2S,5'R)-2-amino-3-(3-carboxy-2-isoxazolin-5-yl)propanoic acid and its 5'S diastereomer, 2-isoxazoline-3-carboxylic acid, and 2-isoxazoline-3,5-dicarboxylic acid
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
thiol compound
-
2,3-dimercaptopropan-1-ol is most effective; required for activity
thiol compound
-
required for activity
additional information
-
no pyridoxal phosphate requirement
-
additional information
-
pyridoxal phosphate does not stimulate
-
additional information
-
no pyridoxal phosphate requirement; one, but not both, of the proton acceptor sites is a thiol
-
KM VALUE [mM]
KM VALUE [mM] Maximum
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.16
-
LL-diaminoheptanedioate
-
-
6.7
-
LL-diaminoheptanedioate
-
at 37°C
0.07
-
meso-diaminoheptanedioate
-
mutant C87S, pH 7.5, 30°C
0.166
-
meso-diaminoheptanedioate
-
wild-type, pH 7.5, 30°C
0.215
-
meso-diaminoheptanedioate
-
mutant C226S, pH 7.5, 30°C
0.36
-
meso-diaminoheptanedioate
-
-
0.36
-
meso-diaminoheptanedioate
-
LL-diaminoheptanedioate
12
17
meso-diaminoheptanedioate
-
recombinant DapF consisting of silent mutation of the first 10 codons of the open reading frame
100
-
meso-diaminoheptanedioate
-
at 37°C
TURNOVER NUMBER [1/s]
TURNOVER NUMBER MAXIMUM[1/s]
SUBSTRATE
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
128
-
LL-2,6-Diaminoheptanedioate
-
forward reaction
84
-
LL-diaminoheptanedioate
-
-
0.001
-
meso-diaminoheptanedioate
-
mutant C87S, pH 7.5, 30°C
0.0025
-
meso-diaminoheptanedioate
-
mutant C226S, pH 7.5, 30°C
0.1465
-
meso-diaminoheptanedioate
-
wild-type, pH 7.5, 30°C
67
-
meso-diaminoheptanedioate
-
-
IC50 VALUE [mM]
IC50 VALUE [mM] Maximum
INHIBITOR
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
IMAGE
0.5
-
dilithium (2Z,6S)-2,6-diamino-4-oxohept-2-enedioate
-
competitive, IC50: 0.5 mM
SPECIFIC ACTIVITY [µmol/min/mg]
SPECIFIC ACTIVITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
0.205
-
-
crude extract after expression
0.574
-
-
after immobilized metal-affinity chromatography
1.18
-
-
after gel filtration
additional information
-
-
-
pH OPTIMUM
pH MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6.5
8
-
in both directions
7.5
-
-
maximum activity of recombinant DapF consisting of silent mutation of the first 10 codons of the open reading frame
pH RANGE
pH RANGE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
6
8.5
-
pH 6: about 60% of maximal activity, pH 8.5: about 25% of maximal activity
6.5
9
-
recombinant DapF consisting of silent mutation of the first 10 codons of the open reading frame
TEMPERATURE OPTIMUM
TEMPERATURE OPTIMUM MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30
-
-
recombinant DapF consisting of silent mutation of the first 10 codons of the open reading frame is almost 50% more active at 30°C than at 25°C
45
-
-
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
TEMPERATURE MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
25
45
-
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 ACCESSION NO.
COMMENTARY
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
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)
MOLECULAR WEIGHT
MOLECULAR WEIGHT MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
30000
-
-
molecular weight of recombinant DapF consisting of silent mutation of the first 10 codons, determined by SDS-PAGE
31030
-
-
predicted, confirmed by SDS-PAGE
45400
-
-
gel filtration
SUBUNITS
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
monomer
-
1 * 34000, SDS-PAGE
Crystallization/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
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
-, Q9LFG2
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
-
sitting drop vapour diffusion method, 2.5 A resolution
-
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
-
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
-
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
-
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
-
pH STABILITY
pH STABILITY MAXIMUM
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
5.5
-
-
2°C, overnight, 0.1 mM 2,3-dimercaptopropan-1-ol, 20% loss of activity
6
-
-
irreversible loss of activity at pH 6 and below. Partially reactivated by thiols
8
-
-
rapid loss of activity when stored in absence of dithiothreitol
8.5
-
-
2°C, overnight, 0.1 mM 2,3-dimercaptopropan-1-ol, 70% loss of activity
GENERAL STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
air oxidation, in absence of reducing thiols, is slower at pH 7 than at pH 8
-
denatured on freezing
-
STORAGE STABILITY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
0-4°C, best storage temperature
-
Purification/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
by immobilized nickel metal-affinity chromatography using 5 ml HisTrap HP columns and by gel filtration as His-tagged enzyme
-
by using nickel-chelate affinity chromatography
-
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
-
partial
-
Cloned/COMMENTARY
ORGANISM
UNIPROT ACCESSION NO.
LITERATURE
expressed in Escherichia coli BL21 (DE3)
-
DAP epimerase mutants C73S and C217S from Haemophilus influenzae are cloned and purified
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expression in Escherichia coli BL21(DE3)
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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 ACCESSION NO.
COMMENTARY
LITERATURE
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
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mutant enzyme is inactive as epimerase, slow elimination of HF from D,L-3-fluoro-2,6-diaminopimelate and L,L-3-fluoro-2,6-diaminopimelate
C73S/C217S
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in order to prevent C73 and C217 of DAP epimerase from oxidation to a disulfide prior to crystallization, DAP epimerase mutants C73S and C217S from Haemophilus influenzae are generated by site-directed mutagenesis
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
additional information
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a recombinant DapF is generated consisting of silent mutation of the first 10 codons of the open reading frame. single nucleotide substitutions are incorporated without changing product composition in the first 30 nucleotides of the dapF open reading frame,in order to disrupt any secondary structure-promoting sequences present. this significantly increases the yield of the enzyme
APPLICATION
ORGANISM
UNIPROT ACCESSION NO.
COMMENTARY
LITERATURE
analysis
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a high-performance liquid chromatography method for the simultaneous assay of diaminopimelate epimerase and decarboxylase
analysis
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simple and sensitive spectrophotometric method for the determination of meso-alpha,epsilon-diaminopimelate with meso-2,6-diaminopimelate D-dehydrogenase and its application to the assay of diaminopimelate epimerase
drug development
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DAP epimerase is an attractive target for rational antibiotic design
drug development
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diaminopimelate epimerase catalyzes the stereoinversion of LL-diaminopimelate to meso-diaminopimelate, a precursor of L-lysine and an essential component of the bacterial peptidoglycan. This function is vital to bacteria and the enzyme therefore represents an attractive target for the design of novel anti-bacterials
drug development
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bacterial racemase, including glutamate racemase and DAP epimerase, are potential targets for the development of new agents effective against organisms resistant to conventional antibiotics
drug development
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enzyme represents a promising target for rational drug design aimed to develop new selective antibacterial therapeutic agents
drug development
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the recombinant DapF produced is correctly folded and is a suitable tool for a drug development study