Information on EC 1.14.11.55 - ectoine hydroxylase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
1.14.11.55
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RECOMMENDED NAME
GeneOntology No.
ectoine hydroxylase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
ectoine + 2-oxoglutarate + O2 = 5-hydroxyectoine + succinate + CO2
show the reaction diagram
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Glycine, serine and threonine metabolism
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SYSTEMATIC NAME
IUBMB Comments
ectoine,2-oxoglutarate:oxygen oxidoreductase (5-hydroxylating)
Requires Fe2+ and ascorbate. The enzyme, found in bacteria, is specific for ectoine.
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
ectD gene optimized for the expression in Escherichia coli
JN019030
GenBank
Manually annotated by BRENDA team
ectD gene optimized for the expression in Escherichia coli
JN019031
GenBank
Manually annotated by BRENDA team
ectD gene optimized for the expression in Escherichia coli
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
ectoine + 2-oxoglutarate + O2
5-hydroxyectoine + succinate + CO2
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
ectoine + 2-oxoglutarate + O2
5-hydroxyectoine + succinate + CO2
show the reaction diagram
additional information
?
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ascorbate
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5 mM, 70% inhibition
Fe2+
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stimulation at 1 mM, inhibitory above
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.7 - 6.2
2-oxoglutarate
2.6 - 19.6
ectoine
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.2 - 8.9
ectoine
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12 - 1.44
ectoine
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3.06
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pH 7.5, 32C
20
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pH not specified in the publication, temperature not specified in the publication
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9.6
6.5 - 9.6
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 50
10 - 47
JN019030
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15 - 45
JN019031
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pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.7
JN019031
calculated
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
36000
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gel filtration
70730
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light scattering, recombinant Strep-tagged protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
monomer
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1 * 34000, SDS-PAGE, 1 * 34400, mass spectrometry
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
to 2.4 A resolution, space group P1, with unit-cell parameters a 45.18 A, b 58.87 A, c 68.81 A, alpha 77.48 degrees, beta 86.03 degrees, gamma 66.97 degrees. The asymmetric unit contains two molecules with a Mattews coefficient of about 2.44 A3/Da and a solvent content of 49.53%
apo-enzyme crystallizes in space group C2221, the iron-supplemented form displays a P212121 symmetry. The apo form contains one monomer per asymmetric unit whereas the Fe-supplemented form contains a dimer
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crystal structure in its apo-form, in complex with iron, and in complex with iron, cosubstrate 2-oxoglutarate, and 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine is bound by residues residues His144, His245, and Asp146 forming the 2-His-1-carboxylate facial triad
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crystal structure in complex with Fe3+ at a resolution of 1.85 A. The core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active site is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate cosubstrate-binding site
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structural comparison, molecular dynamics simulations, and site-directed mutagenesis suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine
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to 1.9 A resolution, and comparison of iron-bound and apo structure. The iron ligand is bound via interaction with histidine side-chains His146 and His248, and the side-chain of Asp-148. These residues form a conserved H6D/EH motif, the so-called 2-His-1-carboxylate facial triad
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
ectoine production is linearly correlated with the salinity of the growth medium. The formation of 5-hydroxyectoine is primarily a stationary growth phase phenomenon. The transcript levels of the ectABC and ectD genes increase as a function of salinity
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E140A
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residue involved in dimerization, activity similar to wild-type
Q127A
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residue involved in ectoine binding, about 1% of wild-type activity
R139A
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residue involved in dimerization, activity similar to wild-type
R139A/E140A
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residues involved in dimerization, activity similar to wild-type
R280A
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residue involved in ectoine binding, about 8% of wild-type activity
T149A
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residue involved in ectoine binding, about 4% of wild-type activity
W150A
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residue involved in ectoine binding, about 2% of wild-type activity
E140A
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residue involved in dimerization, activity similar to wild-type
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Q127A
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residue involved in ectoine binding, about 1% of wild-type activity
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R139A
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residue involved in dimerization, activity similar to wild-type
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R280A
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residue involved in ectoine binding, about 8% of wild-type activity
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T149A
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residue involved in ectoine binding, about 4% of wild-type activity
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A163C
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residue is not involved in ligand binding
A163C/S244C
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residues are not involved in ligand binding
D148A
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loss of activity. Residue is involved in binding of Fe2+
D148E
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loss of activity. Residue is involved in binding of Fe2+
F143A
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loss of activity. Residue is involved in binding of 2-oxoglutarate
F143W
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loss of activity. Residue is involved in binding of 2-oxoglutarate
F143Y
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loss of activity. Residue is involved in binding of 2-oxoglutarate
F242A
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loss of activity. Residue is involved in binding of ectoine
F242W
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loss of activity. Residue is involved in binding of ectoine
F242Y
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3fold increase in Km value
F263A
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loss of activity. Residue is involved in binding of ectoine
F263W
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loss of activity. Residue is involved in binding of ectoine
F263Y
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30% increase in Km value
F95A
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loss of activity. Residue is involved in binding of 2-oxoglutarate
H146A
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loss of activity. Residue is involved in binding of Fe2+
H248A
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loss of activity. Residue is involved in binding of Fe2+
N133A
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loss of activity. Residue is involved in binding of 2-oxoglutarate
N261A
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residue is not involved in ligand binding
P198A
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activity similar to wild-type, residue of loop region
Q129A
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loss of activity. Residue is involved in binding of ectoine
R131A
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loss of activity. Residue is involved in binding of 2-oxoglutarate
R259A
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loss of activity. Residue is involved in binding of 2-oxoglutarate
R259H
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loss of activity. Residue is involved in binding of 2-oxoglutarate
R259K
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loss of activity. Residue is involved in binding of 2-oxoglutarate
R259Q
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loss of activity. Residue is involved in binding of 2-oxoglutarate
S165A
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3fold increase in Km value
S167A
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residue is not involved in ligand binding
S205A
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activity similar to wild-type, residue of loop region
S244C
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residue is not involved in ligand binding
S250A
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loss of activity. Residue is involved in binding of 2-oxoglutarate
V265A
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residue is not involved in ligand binding
V265L
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residue is not involved in ligand binding
V265T
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residue is not involved in ligand binding
W152A
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loss of activity. Residue is involved in binding of ectoine
W152F
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loss of activity. Residue is involved in binding of ectoine
W152Y
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loss of activity. Residue is involved in binding of ectoine
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
synthesis