Information on EC 2.5.1.55 - 3-deoxy-8-phosphooctulonate synthase

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

EC NUMBER
COMMENTARY hide
2.5.1.55
-
RECOMMENDED NAME
GeneOntology No.
3-deoxy-8-phosphooctulonate synthase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
phosphoenolpyruvate + D-arabinose 5-phosphate + H2O = 3-deoxy-D-manno-octulosonate 8-phosphate + phosphate
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
condensation
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
CMP-3-deoxy-D-manno-octulosonate biosynthesis
-
-
CMP-8-amino-3,8-dideoxy-D-manno-octulosonate biosynthesis
-
-
Lipopolysaccharide biosynthesis
-
-
Metabolic pathways
-
-
CMP-KDO biosynthesis
-
-
SYSTEMATIC NAME
IUBMB Comments
phosphoenolpyruvate:D-arabinose-5-phosphate C-(1-carboxyvinyl)transferase (phosphate-hydrolysing, 2-carboxy-2-oxoethyl-forming)
-
CAS REGISTRY NUMBER
COMMENTARY hide
9026-96-4
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
-
-
-
Manually annotated by BRENDA team
AtkdsA2
-
-
Manually annotated by BRENDA team
AtkdsA2
-
-
Manually annotated by BRENDA team
var. italica
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
strain 6BC
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
Gram-negative bacteria
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
Uniprot
Manually annotated by BRENDA team
no activity in Malus sp.
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
SwissProt
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
-
KDO8PS is evolutionarily and structurally related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 2.5.1.54), which uses erythrose 4-phosphate in place of arabinose 5-phosphate. Both KDO8PS and type Ibeta DAH7PS enzymes adopt similar homotetrameric associations with their active sites close to one of the interfaces
malfunction
-
Arabidopsis thaliana kdsA male gametophyte with lethal phenotype; Arabidopsis thaliana kdsA male gametophyte with lethal phenotype; while single T-DNA mutants of either gene display no visible phenotypes, generation of a double knockout mutant has not been possible due to a failure of haploid pollen tube elongation; while single T-DNA mutants of either gene display no visible phenotypes, generation of a double knockout mutant has not been possible due to a failure of haploid pollen tube elongation
metabolism
-
catalyzes the formation of 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P), a key precursor in the biosynthesis of the endotoxin of Gram-negative bacteria
physiological function
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
phosphoenolpyruvate + 2-deoxy-D-ribose 5-phosphate
? + phosphate
show the reaction diagram
phosphoenolpyruvate + 2-deoxyribose 5-phosphate
?
show the reaction diagram
-
-
-
-
?
phosphoenolpyruvate + D-arabinose 5-phosphate
2-dehydro-3-deoxy-D-octonate 8-phosphate + phosphate
show the reaction diagram
phosphoenolpyruvate + D-arabinose 5-phosphate + H2O
2-dehydro-3-deoxy-D-octonate 8-phosphate + phosphate
show the reaction diagram
phosphoenolpyruvate + D-arabinose 5-phosphate + H2O
3-deoxy-D-manno-octulosonate 8-phosphate + phosphate
show the reaction diagram
phosphoenolpyruvate + D-arabinose-5-phosphate + H2O
2-dehydro-3-deoxy-D-octonate 8-phosphate + phosphate
show the reaction diagram
phosphoenolpyruvate + erythrose 4-phosphate
3-deoxy-D-ribo-heptulosonate 7-phosphate
show the reaction diagram
phosphoenolpyruvate + L-xylose 5-phosphate + H2O
?
show the reaction diagram
-
-
-
?
phosphoenolpyruvate + ribose 5-phosphate
?
show the reaction diagram
additional information
?
-
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
phosphoenolpyruvate + D-arabinose 5-phosphate + H2O
2-dehydro-3-deoxy-D-octonate 8-phosphate + phosphate
show the reaction diagram
phosphoenolpyruvate + D-arabinose 5-phosphate + H2O
3-deoxy-D-manno-octulosonate 8-phosphate + phosphate
show the reaction diagram
phosphoenolpyruvate + D-arabinose-5-phosphate + H2O
2-dehydro-3-deoxy-D-octonate 8-phosphate + phosphate
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ca2+
-
no activation of wild type enzyme and metal-dependent mutants, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4
Fe3+
-
1 mM, 5-10% stimulation
Li+
-
1 mM, 5-10% stimulation
Magnesium
-
wild-type enzyme contains 0.05 mol of magnesium per mol of enzyme, mutant enzyme N26C contains 0.4 mol of magnesium per mol of enzyme
Ni2+
-
no activation of wild type enzyme and metal-dependent mutants, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4
Sr2+
-
no activation of wild type enzyme and metal-dependent mutants, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(Z,E)-D-Glucophosphoenolpyruvate
Gram-negative bacteria
-
and its carboxylic ester derivatives
1,10-phenanthroline
1-carboxyheptane-1,7-diyl bis(phosphate)
2,6-Anhydro-3-deoxy-2beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate
-
most potent inhibitor
2,6-pyridine dicarboxylic acid
-
IC50: 0.0422
2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[[(1R,2R,3S,4S,5R)-2,3,4-trihydroxy-5-(hydroxymethyl)cyclohexyl]oxy]-4H-chromen-4-one
i.e. hyperin, structure analysis, and enzyme interaction study, binding structure, overview
2-dehydro-3-deoxy-D-octonate 8-phosphate
-
mutant enzyme R106G is more severely inhibited than wild-type enzyme
2-Deoxy-2-fluoro-D-arabinoate-5-phosphate
-
-
6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine
-
PD404,182,displays strong inhibition in vitro, but is ineffective in vivo against Gram-negative bacteria due to an inability to cross the bacterial plasma membran
-
API
arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) bisubstrate inhibitor
-
Bromopyruvate
-
phosphoenolpyruvate, but not arabinose-5-phosphate protects
D-arabinose 5-phosphate
-
mutant enzyme R106G is less severely inhibited than wild-type enzyme
D-ribose 5-phosphate
dipicolinic acid
-
N-[3-(furan-2-yl)phenyl]-1-(3-phenylpropyl)piperidine-4-carboxamide
i.e. MC181, structure analysis, and enzyme interaction study, binding structure, overview
phosphate
additional information
21 inhibitor compounds synthesis and screening based on the API inhibitor structure, docking study and molecular modelling, overview
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.23 - 5
2-deoxy-D-ribose 5-phosphate
0.05
2-deoxyribose 5-phosphate
-
pH 7.6, 37°C
0.0005 - 3.7
D-arabinose 5-phosphate
0.057
L-xylose 5-phosphate
-
at 30°C
0.0000066 - 1.65
phosphoenolpyruvate
additional information
additional information
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.007 - 0.66
2-deoxy-D-ribose 5-phosphate
0.12
2-deoxyribose 5-phosphate
Escherichia coli
-
pH 7.6, 37°C
0.01 - 9
D-arabinose 5-phosphate
1.1
L-xylose 5-phosphate
Neisseria meningitidis
-
at 30°C
0.02 - 100
phosphoenolpyruvate
additional information
additional information
Neisseria meningitidis
-
N23C/C246S/D247E mutant control is barely active, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA; N23C/C246S/D247E/P249A mutant control is barely active, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA; N23C/C246S/P249A mutant control is barely active, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.016 - 0.57
2-deoxy-D-ribose 5-phosphate
1173
0.016 - 1400
D-arabinose 5-phosphate
538
0.4 - 3200
phosphoenolpyruvate
51
additional information
additional information
Neisseria meningitidis
-
N23C/C246S/D247E mutant control is barely active, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA; N23C/C246S/D247E/P249A mutant control is barely active, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA; N23C/C246S/P249A mutant control is barely active, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0079 - 0.02
1-carboxyheptane-1,7-diyl bis(phosphate)
0.6
2,6-Anhydro-3-deoxy-2beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate
-
pH 7.3, 37°C
0.000026
6H-6-Imino-(2,3,4,5-tetrahydropyrimido)[1,2-c]-[1,3]benzothiazine
-
pH and temperature not specified in the publication
-
1
D-ribose 5-phosphate
-
pH 7.3, 37°C
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0293
1,10-phenanthroline
Helicobacter pylori
-
IC50: 0.0293 mM
0.0422
2,6-pyridine dicarboxylic acid
Helicobacter pylori
-
IC50: 0.0422 mM
0.212
EDTA
Helicobacter pylori
-
IC50: 0.212 mM
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.01
-
N23C/C246S/D247E mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA; N23C/C246S/D247E/P249A mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
0.06
-
N23C/C246S/P249A mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
0.19
-
N23C/C246S mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
0.3
-
N23C/C247E mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
0.35
-
N23C/C247E/P249A mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
0.6
-
crude cell lysate, at 25°C and pH 7.5
0.76
-
N23C/P249A mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
1.9
-
N23C mutant control, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
2
-
purified enzyme, at 25°C and pH 7.5
2.34
-
N23C/P249A mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
2.9
-
N23C mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
3.1
-
N23C/C247E mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
4.34
-
N23C/C247E/P249A mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
5
-
N23C/C246S mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
5.64
-
N23C/C246S/D247E/P249A mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
6.3
-
D247E/P249A mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
6.39
-
D247E mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
8.4
-
N23C/C246S/D247E mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
9.8
-
N23C/C246S/P249A mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 90 microM MnSO4.H2O
10.1
-
C246S mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
11.7
-
P249A mutant, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
12
-
wild type, 200 microM phosphoenolpyruvate, 37°C, 50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.4, 20 microM EDTA
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4 - 6
-
and a second optimum at pH 9.0
5.5 - 6
-
in 10-min reaction assay
7.4
-
50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer
9
-
and a second optimum at pH 4.0-6.0
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.8 - 9.15
-
50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, wild type enzyme is most active between pH 6.8 and pH 8.1
4.5 - 7.5
-
pH 4.5: about 50% of maximal activity, pH 7.5: about 50% of maximal activity
5.5 - 10
-
pH 5.5: about 35% of maximal activity, pH 10.0: about 65% of maximal activity, at 50°C
6.1 - 9
-
pH 6.1: about 35% of maximal activity, pH 9.0: about 25% of maximal activity
6.5 - 9.5
-
broad peak between pH 6.5 and 9.5 showing 90% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
-
assay at; assay at
80
-
in 10-min reaction assay
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 70
-
50 mM 1,3-bis[tris(hydroxymethyl)-methylamino]propane (BTP) buffer, pH 7.5, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 60°C, 70°C
37 - 61
-
37°C: about 40% of maximal activity, 61°C: about 55% of maximal activity
45 - 70
-
45°C: about 50% of maximal activity, 70°C: about 55% of maximal activity
60 - 90
-
60°C: about 30% of maximal activity, 70°C: about 70% of maximal activity, 90°C: about 40% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LekdsA mRNAs are preferentially expressed in dividing tissues during fruit development
Manually annotated by BRENDA team
in fulla differentiated leaves the basal activity is 1.5fold lower than in immature leAVES
Manually annotated by BRENDA team
AtkdsA1 is predominantly expressed in shoots
Manually annotated by BRENDA team
additional information
isoform AtkdsA2 gene expression is preferentially associated with plantlet organs displaying a meristematic activity
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Aquifex aeolicus (strain VF5)
Brucella melitensis biotype 1 (strain 16M / ATCC 23456 / NCTC 10094)
Burkholderia ambifaria (strain MC40-6)
Burkholderia cenocepacia (strain ATCC BAA-245 / DSM 16553 / LMG 16656 / NCTC 13227 / J2315 / CF5610)
Burkholderia pseudomallei (strain 1710b)
Burkholderia pseudomallei (strain 1710b)
Burkholderia pseudomallei (strain 1710b)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Escherichia coli (strain K12)
Haemophilus influenzae (strain ATCC 51907 / DSM 11121 / KW20 / Rd)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Neisseria meningitidis serogroup B (strain MC58)
Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)
Vibrio cholerae serotype O1 (strain ATCC 39541 / Classical Ogawa 395 / O395)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
29348
-
x * 29348, calculation from nucleotide sequence
29736
-
3 * 29736, electrospray ionization mass spectrometry
30400
-
monomer with 1 Co2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
30440
-
monomer with 1 Zn2+ or 1 Cu2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
30480
-
monomer with 1 Cd2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
30842
-
x * 30842, electrospray mass spectrometry
30845
-
1 * 30845, ESI-MS, unbound enzyme with substrate phosphoenolpyruvate or product phosphate favors the formation of monomers, phosphoenolpyruvate-bound and unbound enzyme exists as monomer; 2 * 30845, ESI-MS, predominant in unbound enzyme in complex with substrate D-arabinose 5-phosphate or product 3-deoxy-D-manno-octulosonate 8-phosphate, phosphoenolpyruvate-bound and unbound enzyme exists as dimer; 4 * 30845, ESI-MS, phosphoenolpyruvate-bound enzyme exists as tetramer to a low extent, unbound enzyme does not exist in tetrameric state, phosphoenolpyrovate stabilizes the tetrameric structure and may bind at the same position as phosphate
30850
-
monomer, ESI-MS
31519
-
2 * 31519, MALDI-MS
32000
-
3 * 32000, SDS-PAGE
60690
-
dimer with 1 Cd2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
60760
-
dimer with 1 Co2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
60840
-
dimer with 2 Zn2+ or 2 Cu2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
62680
-
dimer, ESI-MS
76000
-
gel filtration
90000
-
gel filtration, non-denaturing PAGE
97000
-
wild-type enzyme, gel filtration
97600
-
gel filtration
121500
-
tetramer with 2 Co2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
121600
-
tetramer with 4 Zn2+ or 4 Cu2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
121800
-
tetramer with 4 Cd2+, ESI-MS, 50 mM ammonium acetate solution, pH 7.5
123400
-
tetramer, ESI-MS
130000
-
native protein, gel-filtration chromatography
additional information
-
two poorly resolved peaks are detected by gel filtration of the mutant enzyme P145S: 91000 Da and 53000 Da
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
monomer
-
1 * 30845, ESI-MS, unbound enzyme with substrate phosphoenolpyruvate or product phosphate favors the formation of monomers, phosphoenolpyruvate-bound and unbound enzyme exists as monomer
oligomer
-
the enzyme structure is a prototypical alpha/beta TIM barrel structure expected from this family of enzymes and contains a tetramer in the asymmetric unit
tetramer
trimer
additional information
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
data reading at -173°C
-
structure of the metal-free and Cd2+ forms of the enzyme are determined in the uncomplexed state and in complex with various combinations of phosphoenolpyruvate, arabinose 5-phosphate and erythrose 4-phosphate
-
X-ray structure of wild-type enzyme shows that when both phosphoenolpyruvate and D-arabinose 5-phosphate bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with phosphoenolpyruvate and phosphoenolpyruvate plus D-arabinose 5-phosphate bound reveal that in R106G closure of the L7 loop is impaired
-
purified recombinant enzyme, hanging-drop vapor diffusion method, mixing of 300 nl of 10 mg/ml protein in 30 mM Tris-HCl, pH 8.0, 200 mM NaCl with 300 nl reservoir solution containing 28% v/v PEG 400, 0.1 M HEPES, pH 7.5, and 0.2 M CaCl2, X-ray diffraction structure determinatin and analysis; purified recombinant enzyme, hanging-drop vapor diffusion method, mixing of 300 nl of 10 mg/ml protein in 30 mM Tris-HCl, pH 8.0, 200 mM NaCl with 300 nl reservoir solution containing 28% v/v PEG 400, 0.1 M HEPES, pH 7.5, and 0.2 M CaCl2, X-ray diffraction structure determinatin and analysis
-
crystallization is performed by vapor diffusion in hanging drops
-
crystals are grown at 23°C by vapor diffusion in hanging drops, crystal structures of the enzyme in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor
-
structures of apo-enzyme and of binary complexes with the substrates phosphoenolpyruvate, the product 2-dehydro-3-deoxy-D-octonate 8-phosphate and the catalytically inactive 1-deoxy analog of arabinose 5-phosphate
-
purified recombinant His-tagged wild-type and mutant H204A enzymes in apoform, or wild-type enzyme complexed with Zn2+ or Cd2+, hanging drop vapor diffusion method, mixing of 0.001 ml of protein solution, containing inhibitor in a 1:2 ratio, with 0.001 ml of reservoir solution, containing 18% PEG 3350, 0.1 M HEPES, pH 7.5, and 0.25 M magnesium chloride hexahydrate, equilibration against reservoir solution, 20°C, 14 days, X-ray diffraction structure determination and analysis at 2.0 A resolution, molecular replacement and modelling
purified recombinant mutant enzymes, hanging-drop vapor diffusion, mixing of 0.002 ml of protein solution containing 20 mg/ml protein in 10 mM BTP, pH 7.5, with 0.002 ml of reservoir solution containing 100 mM sodium acetate, pH 4.6, and 0.6-3.0 M NaCl, equilibration against 0.5 ml reservoir solution, 20°C, 24 h, X-ray diffraction structure determination and analysis at 1.75-2.10 A resolution
-
the X-ray structures, determined for the mutants with altered KANRS motif, indicate no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlate with their altered catalytic function
-
vapor-diffusion hanging drop method, pH 4.6 and pH 5.0, no diffraction quality crystals at higher pH in the range of the active enzyme
-
purified recombinant His6-tagged enzyme, sitting drop vapour diffusion method, mixing of 0.003 ml of 5 mg/ml protein in 0.2 M Tris-HCl, pH 7.4, 0.1 M NaCl, 0.05% 2-mercaptoethanol, and 0.1 mM phosphoenolpyruvate with 0.0015 ml of reservoir solution containing 0.2 M calcium acetate, 0.1 M sodium cacodylate, pH 6.5, 18% w/v PEG 8000, 20°C, 2 days, X-ray diffraction structure determination and analysis at 2.8 A resolution, molecular replacementusing structure PDB ID 1g7v and model building
-
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27
-
unstable in absence of phosphoenolpyruvate
40
-
30 min, in absence of any substrate or in presence of D-arabinose 5-phosphate, 30% loss of activity, no inactivation in presence of phosphoenolpyruvate
42
-
half-life of enzyme from mutant strain PR32, without D-arabinose-5-phosphate: 2.5 min. Half-life of enzyme from mutant strain PR32, in presence of 13 mM D-arabinose 5-phosphate: 9.5 min. Half-life of enzyme from parent strain PR122: 13 min
45
-
15 min, 30% loss of activity
60
-
20% incrase in activity after 7 min
68
-
mutant C21N, presence of Cd2+, melting temperature; mutant D243E, melting temperature; wild-type, melting temperature
69
-
mutant C21N, melting temperature
71
-
mutant P245A, melting temperature
78
-
mutant D243E, presence of Cd2+, melting temperature
79
-
mutant P245A, presence of Cd2+, melting temperature
82
-
wild-type, presence of Cd2+, melting temperature
additional information
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
Cd2+ destabilizes the enzyme, overview
-
dialysis against 0.01 M Tris-HCl, pH 7.5, containing 0.01 M thioglycerol for 4 h, less than 20% loss of activity
-
dialysis against 10 mM EDTA in 50 mM Tris-HCl buffer, pH 7.3, inactivates
-
repeated freezing and thawing causes loss of activity
-
substrate phosphoenolpyruvate stabilizes the wild-type and mutant enzymes
-
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 50% loss of activity after 14 days
-
-90°C, 0.1 M potassium phosphate buffer, pH 7.2, stable for up to 1 year
-
4°C, in 20 mM ammonium acetate (pH 7.8), overnight, remains stable in the phosphoenol-bound state
-
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
apoenzyme + 200 microM metal ion salt + 50 mM Tris-HCl buffer, pH 7.5 dialyzed against 50 mM ammonium acetate buffer, pH 7.5, 4°C
-
enzyme purification followed by incubation with 50 microM Cd2+, Zn2+, or Cu2+ in 20 mM Tris-HCl buffer, pH 7.5, to substitute the metal ions Fe2+ or Co2+
-
one-step purification
-
overnight dialysis against 50 mM ammonium acetate, pH 7.8, 4°C, reconstitution with substrates and products
-
recombinant enzyme
recombinant enzyme from wild-type strain AG701 and temperature-sensitive strain AG701i50
-
recombinant His-tagged enzyme from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and ultrafiltration; recombinant His-tagged enzyme from Escherichia coli strain BL21 (DE3) by nickel affinity chromatography and ultrafiltration
-
recombinant His-tagged enzyme in Escherichia coli strain pTf16/BL21 by nickel affinity chromatography, dialysis, and ultrafiltration
recombinant His6-tagged enzyme from Escherichia coli strain Rosetta2 (pLysS) DE3 by nickel affinity chromatography
-
Source 15Q column chromatography and Source 15Phe column chromatography
-
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
1 15.5-kb segment containing the kdsA gene, expression in Escherichia coli
-
cloned into a T7-driven expression vector, overexpression in Escherichia coli
-
enzyme from wild-type strain AG701 and temperature-sensitive strain AG701i50 expressed in Escherichia coli
-
expressed in Escherichia coli
-
expressed in Escherichia coli BL21(DE3) cells
-
expression in Escherichia coli
expression in Escherichia coli BL21(DE3)
-
expression in Salmonella enterica serovar typhimurium
expression in Salmonella enterica serovar typhimurium AG701i50
-
expression in Salmonella typhimurium
-
gene kdo8ps, recombinant expression of His6-tagged codon-optimized enzyme in Escherichia coli strain Rosetta2 (pLysS) DE3
-
gene kdsA, recombinant expression of His-tagged enzyme in Escherichia coli strain pTf16/BL21
gene kdsA1, in Arabidopsis a functionally redundant gene pair encodes Kdo-8-P synthases, AtKdsA1 (At1g9500) and AtKdsA2 (At1g16340), recombinant expression in Escherichia coli; gene kdsA2, in Arabidopsis a functionally redundant gene pair encodes Kdo-8-P synthases, AtKdsA1 (At1g9500) and AtKdsA2 (At1g16340), recombinant expression in Escherichia coli
-
gene kdsA1, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 (DE3); gene kdsA2, recombinant expression of His-tagged enzyme in Escherichia coli strain BL21 (DE3)
-
in Escherichia coli with plasmid pet28a
-
localization of the kdsA gene with the aid of the physical map of the Escherichia coli chromosome
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A56P/N57DELTA
-
mutation in the absolutely conserved KANRS motif. Complete loss of activity
C21N
-
complete loss of activity
D243A
-
mutation of the absolutely conserved 243AspGlyPro245 motif, complete loss of activity
D243Q
-
mutation of the absolutely conserved 243AspGlyPro245 motif, active enzyme with altered metal-dependency
K55A
-
mutation in the absolutely conserved KANRS motif. Complete loss of activity
N57A
-
mutation in the absolutely conserved KANRS motif. About 2% of wild-type activity
N57DELTA
-
mutation in the absolutely conserved KANRS motif. Complete loss of activity
P245A
-
mutation of the absolutely conserved 243AspGlyPro245 motif, active enzyme with altered metal-dependency
C11N
-
mutant is not capable of binding metal and lacks the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, shows decreased thermal stability
C11N/S235P/Q237A
-
mutant is not capable of binding metal and lacks the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, shows decreased thermal stability
H185G
-
mutation decreases the affinity of the enzyme to bind Fe2+, but not Zn2+. Maximal activity, about 8-10% of the wild-type activity is obtained when the native metal is replaced with Cd2+
P10M/C11N
-
mutant is not capable of binding metal and lacks the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, shows decreased thermal stability
P10M/C11N/S235P/Q237A
-
mutant is not capable of binding metal and lacks the structural asymmetry among subunits with regard to substrate binding and conformation of the L7 loop, shows decreased thermal stability
R106G
-
the closure of the L7 loop is impaired. The mutant enzyme shows a smaller KM-value for phosphoenolpyruvate, larger Ki-value and KM-value for D-arabinose 5-phosphate and smaller Ki-values for phosphate and 2-dehydro-3-deoxy-D-octonate 8-phosphate compared ti wild-type enzyme
C11A
-
mutant enzyme retains less than 1% of the wild-type activity and is incapable of metal binding. Activity is not stimulated by Mn2+, Co2+ and Zn2+. Cd2+ stimulates 2fold at a concentration above 1 mM
C11N
enzyme retains 10% of the wild-type activity in absence of metal ions. Addition of divalent metal ions does not affect the catalytic activity of the mutant enzyme and the catalytic efficiency, i.e. the ratio of turnover number to Km-value, is reduced only 12fold, the mutant enzyme has become metal-independent
N26C
-
activity of the wild-type enzyme is independent of metal ions, the activity of mutant enzyme is decreased by EDTA and increased by Mn2+ and Cd2+
A56P/N57DELTA
-
mutation in the absolutely conserved KANRS motif. Complete loss of activity
F114A
-
site-directed mutagenesis, conversion to the corresponding residue in enzyme DAH7PS, EC 2.5.1.54, the mutant shows altered kinetics compared to the wild-type, structure analysis
F114R
-
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type, structure analysis
F114R/R117A
-
site-directed mutagenesis, the mutant shows altered kinetics compared to the wild-type, structure analysis
F114R/R117Q
-
site-directed mutagenesis, conversion to the corresponding residue in enzyme DAH7PS, EC 2.5.1.54, the mutant shows altered kinetics compared to the wild-type, structure analysis
F114R/R117Q/F139G
-
site-directed mutagenesis, conversion to the corresponding residue in enzyme DAH7PS, EC 2.5.1.54, the mutant shows altered kinetics compared to the wild-type, structure analysis
F139G
-
site-directed mutagenesis, conversion to the corresponding residue in enzyme DAH7PS, EC 2.5.1.54, the mutant shows altered kinetics compared to the wild-type, structure analysis
K57A
-
mutation in the absolutely conserved KANRS motif. Complete loss of activity
N57D
-
mutation in the absolutely conserved KANRS motif. Complete loss of activity
N59DELTA
-
mutation in the absolutely conserved KANRS motif. Less than 0.1% of wild-type activity
R117K
-
site-directed mutagenesis, conversion to the corresponding residue in enzyme DAH7PS, EC 2.5.1.54, the mutant shows altered kinetics compared to the wild-type, structure analysis
R117Q
-
site-directed mutagenesis, conversion to the corresponding residue in enzyme DAH7PS, EC 2.5.1.54, the mutant shows altered kinetics compared to the wild-type, structure analysis
P145S
-
natural mutation in the temperature-sensitive strain AG701i50 leads to an increase in Km-value for both substrates, D-arabinose 5-phosphate and phosphoenolpyruvate, this mutant enzyme also has an altered oligomeric state. Reduced activity, about 35% of the wild-type, between 15 and 30°C. Above 30°C the activity of the mutant enzyme decreases dramatically
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
medicine
-
enzyme absent in mammals, therefore potential target for the development of new antibiotics
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