Information on EC 4.99.1.5 - aliphatic aldoxime dehydratase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.99.1.5
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RECOMMENDED NAME
GeneOntology No.
aliphatic aldoxime dehydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
an aliphatic aldoxime = an aliphatic nitrile + H2O
show the reaction diagram
; The enzyme from Pseudomonas chlororaphis contains Ca2+ and protoheme IX, the iron of which must be in the form Fe2+ for activity. The enzyme exhibits a strong preference for aliphatic aldoximes, such as butyraldoxime and acetaldoxime, over aromatic aldoximes, such as pyridine-2-aldoxime, which is a poor substrate. No activity was found with the aromatic aldoximes benzaldoxime and pyridine-4-aldoxime
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dehydration
additional information
SYSTEMATIC NAME
IUBMB Comments
aliphatic aldoxime hydro-lyase (aliphatic-nitrile-forming)
The enzyme from Pseudomonas chlororaphis contains Ca2+ and protoheme IX, the iron of which must be in the form Fe(II) for activity. The enzyme exhibits a strong preference for aliphatic aldoximes, such as butyraldoxime and acetaldoxime, over aromatic aldoximes, such as pyridine-2-aldoxime, which is a poor substrate. No activity was found with the aromatic aldoximes benzaldoxime and pyridine-4-aldoxime.
CAS REGISTRY NUMBER
COMMENTARY hide
203210-76-8
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain OxB-1
-
-
Manually annotated by BRENDA team
strain OxB-1
-
-
Manually annotated by BRENDA team
K-9
SWISSPROT
Manually annotated by BRENDA team
K-9
SWISSPROT
Manually annotated by BRENDA team
strain A-4
SwissProt
Manually annotated by BRENDA team
strain A-4
SwissProt
Manually annotated by BRENDA team
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(E)-acetaldoxime
?
show the reaction diagram
-
-
-
-
?
(E)-pyridine-3-aldoxime
pyridine-3-nitrile + H2O
show the reaction diagram
not Z-form, poor substrate, 0.78% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-2-phenylpropionaldoxime
2-phenylpropionitrile + H2O
show the reaction diagram
(E/Z)-2-phenylpropionaldoxime
2-phenylpropiononitrile + H2O
show the reaction diagram
5.23% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-3-phenylpropionaldoxime
3-phenylpropionitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-4-phenybutyraldoxime
4-phenybutyrnitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-acetaldoxime
acetonitrile + H2O
show the reaction diagram
(E/Z)-cyclohexanecarboxaldehyde oxime
?
show the reaction diagram
(E/Z)-cyclohexanecarboxaldehyde oxime
cyclohexanenitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-indoleacetaldoxime
indoleacetonitrile + H2O
show the reaction diagram
7.29% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-isobutyraldoxime
isobutyrnitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-isobutyraldoxime
isobutyronitrile + H2O
show the reaction diagram
26.5% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-isocapronaldoxime
isocaprononitrile + H2O
show the reaction diagram
(E/Z)-isovaleraldoxime
isovalernitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-isovaleraldoxime
isovaleronitrile + H2O
show the reaction diagram
58.6% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-mandelaldoxime
mandelonitrile + H2O
show the reaction diagram
3.09% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-n-butyraldoxime
n-butyrnitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-n-butyraldoxime
n-butyronitrile + H2O
show the reaction diagram
(E/Z)-n-capronaldoxime
n-capronnitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-n-capronaldoxime
n-caprononitrile + H2O
show the reaction diagram
64.2% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-n-valeraldoxime
n-valernitrile + H2O
show the reaction diagram
-
-
-
?
(E/Z)-n-valeraldoxime
n-valeronitrile + H2O
show the reaction diagram
(E/Z)-propionaldoxime
propionitrile + H2O
show the reaction diagram
(E/Z)-propionaldoxime
propiononitrile + H2O
show the reaction diagram
7.76% of the activity with (E/Z)-cyclohexanecarboxaldehyde oxime
-
?
(E/Z)-pyridine-2-aldoxime
pyridine-2-nitrile + H2O
show the reaction diagram
poor substrate
-
?
(Z)-3-phenylpropionaldoxime
3-phenylpropiononitrile + H2O
show the reaction diagram
(Z)-acetaldoxime
?
show the reaction diagram
-
preferred substrate
-
-
?
(Z)-phenylacetaldoxime
phenylacetonitrile + H2O
show the reaction diagram
aldoxime
nitrile + H2O
show the reaction diagram
aliphatic aldoxime
aliphatic nitrile + H2O
show the reaction diagram
alkylaldoxime
alkylnitrile + H2O
show the reaction diagram
an aliphatic aldoxime
an aliphatic nitrile + H2O
show the reaction diagram
butyraldoxime
butyronitrile + H2O
show the reaction diagram
n-butyraldoxime
n-butyronitrile + H2O
show the reaction diagram
propionaldoxime
propionitrile + H2O
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
(E/Z)-isocapronaldoxime
isocaprononitrile + H2O
show the reaction diagram
(E/Z)-n-butyraldoxime
n-butyronitrile + H2O
show the reaction diagram
alkylaldoxime
alkylnitrile + H2O
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
heme b
prosthetic group, protoheme IX, heme iron is present in a reduced form, contains 0.37 mol heme per mol of enzyme
protoheme IX
additional information
no requirement of FMN
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Ag+
inhibitory
Ca2+
contains 1.58 mol Ca2+ per mol of homodimeric OxdA, may act as another cofactor
Cd2+
activates at 0.1 mM, strong inhibition at 1 mM
Cr3+
inhibitory
Cu2+
activates at 0.1 mM, strong inhibition at 1 mM
Pb2+
inhibitory
Zn2+
activates at 0.1 mM, strong inhibition at 1 mM
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis-2-nitrobenzoate
weak inhibition
AgNO3
very sensitive to, concentration-dependent, 1 mM, 85.8% inhibition
Cd2+
strong inhibition at 1 mM, activates at 0.1 mM
Co2+
strong inhibition at 1 mM, activates at 0.1 mM
Cu+
strong inhibition at 1 mM, activates at 0.1 mM
Cu2+
strong inhibition at 1 mM, activates at 0.1 mM
diethyldithiocarbamate
1 mM, 40.6% inhibition
digallol
slight inhibition
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dimethylphenylenediamine
slight inhibition
guaiacol
slight inhibition
heavy metal ion
1 mM
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hydroxylamine
concentration-dependent, 1 mM, 100% inhibition
iodoacetate
weak inhibition
miconazole
slight inhibition
N-ethylmaleimide
weak inhibition
nitroblue tetrazolium
slight inhibition
p-chloromercuribenzoate
weak inhibition
p-phenylenediamine
slight inhibition
phenazine methosulfate
slight inhibition
phenylhydrazine
Sulfhydryl reagent
1 mM
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tetramethylphenylenediamine
slight inhibition
Tiron
inhibits dehydration of (Z)-3-phenylpropionaldoxime, but not of (E/Z)-n-valeraldoxime
trimethylhydroquinone
slight inhibition
Z-phenylacetaldoxime
-
Zn2+
strong inhibition at 1 mM, activates at 0.1 mM
additional information
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2-mercaptoethanol
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 2.13fold
cysteamine
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 3.93fold
duroquinone
activates
FAD
1 mM, 1.2fold activation in presence of Na2S, 4.5fold in absence of Na2S
FMN
1 mM, 1.7fold activation in presence of Na2S, 2.4fold in absence of Na2S
L-cysteine
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 2.39fold
Na2S
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 8.35fold
Na2S2O4
Na2S2O5
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 5.84fold
Na2SO3
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 7.51fold
reducing reagent
requirement, activates
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riboflavin
1 mM, 1.1fold activation in presence of Na2S, 4.2fold in absence of Na2S
thioglycerol
activates, (Z)-phenylacetaldoxime dehydration, 1 mM, 4.06fold
vitamin K3
activates
additional information
not activated by NaHSO3, Na2SO4, NaHSO4 or Na2S2O7
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
20
(E)-pyridine-3-aldoxime
pH 7, 30°C
4.07 - 11.9
(E/Z)-2-phenylpropionaldoxime
0.882
(E/Z)-4-phenybutyraldoxime
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11
(E/Z)-acetaldoxime
pH 7, 30°C
1.13
(E/Z)-cyclohexanecarboxaldehyde oxime
pH 7, 30°C
5.96
(E/Z)-cyclohexanecarboxaldehydeoxime
-
3.91
(E/Z)-indoleacetaldoxime
pH 7, 30°C
0.538 - 5.54
(E/Z)-isobutyraldoxime
6.76
(E/Z)-isocapronaldoxime
pH 7, 30°C
1.33 - 3.97
(E/Z)-isovaleraldoxime
3.23
(E/Z)-mandelaldoxime
pH 7, 30°C
0.25 - 2.16
(E/Z)-n-butyraldoxime
2.94 - 3.12
(E/Z)-n-capronaldoxime
1.13 - 3.78
(E/Z)-n-valeraldoxime
0.778 - 5.13
(E/Z)-propionaldoxime
3.4
(E/Z)-pyridine-2-aldoxime
pH 7, 30°C
0.975 - 2.31
(Z)-3-phenylpropionaldoxime
0.991 - 1.4
(Z)-phenylacetaldoxime
additional information
additional information
Km for the activating Na2S is 0.0408 mM
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
336
(E/Z)-acetaldoxime
Pseudomonas chlororaphis
Q7WSJ4
pH 7, 30°C
324
(E/Z)-n-butyraldoxime
Pseudomonas chlororaphis
Q7WSJ4
pH 7, 30°C
5.4
(E/Z)-pyridine-2-aldoxime
Pseudomonas chlororaphis
Q7WSJ4
pH 7, 30°C
5.1 - 47.3
(Z)-phenylacetaldoxime
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0879
pH 7, 30°C, wild-type OxdRG, (Z)-phenylacetaldoxime as substrate, in presence of 1 mM Na2S
0.758
pH 7, 30°C, butyraldoxime as substrate, under aerobic conditions
2.18
pH 7, 30°C, recombinant OxdRG, (Z)-phenylacetaldoxime as substrate, in presence of 1 mM Na2S
197
pH 7, 30°C, butyraldoxime as substrate, under anaerobic reduced conditions, OxdA reduced by Na2S2O4
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
8
0.1 M potassium phosphate buffer
additional information
maximum at pH 5.5, another high activity level occurs at pH 9.4
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
active over a broad pH-range
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
around, 0.1 M potassium phosphate buffer, pH 8
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
38000
2 * 40127, calculated from the amino acid sequence, 2 * 38000, SDS-PAGE
39892
2 * 42000, wild-type OxdRG, SDS-PAGE, 2 * 39892, calculated from the sequence
40127
2 * 40127, calculated from the amino acid sequence, 2 * 38000, SDS-PAGE
42000
2 * 42000, wild-type OxdRG, SDS-PAGE, 2 * 39892, calculated from the sequence
42500
2 * 42500, gel filtration
44510
sequence
76400
OxdA without reducing conditions, gel filtration
85000
SDS-PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 100 mM CAPSO (pH 9.4), 2.0 M ammonium sulfate, and 12% (v/v) glycerol
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18% (w/v) PEG 4000, 0.075 M sodium cacodylate, 0.1 M magnesium acetate, pH 7.4, vapor diffusion, hanging drop, temperature 293 K, crystal structure of substrate-free form of aldoxime dehydratase, resolution 1.79 A, space group P1211, aldoxime dehydratase (OxdRE) in complex with propionaldoxime (soaked crystal), resolution 1.6 A, aldoxime dehydratase (OxdRE) in complex with butyraldoxime (co-crystal), resolution 2.5 A, aldoxime dehydratase (OxdRE) in complex with butyraldoxime (soaked crystal), resolution 1.8 A
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 6.5
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677719
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
after 15 min preincubation OxdA is reduced by Na2S2O4, under anaerobic conditions, 100% of enzyme activity
25
after 15 min preincubation OxdA is reduced by Na2S2O4, under anaerobic conditions, 94% of enzyme activity
35
after 15 min preincubation OxdA is reduced by Na2S2O4, under anaerobic conditions, 78% of enzyme activity
45
after 15 min preincubation OxdA is reduced by Na2S2O4, under anaerobic conditions, 14% of enzyme activity
50
after 15 min preincubation OxdA is reduced by Na2S2O4, under anaerobic conditions, 3.2% of enzyme activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; by ammonium sulfate fractionation and DEAE-Sephacel, TSK gel Butyl-Toyopearl 650M and Cellulofine HAp column chromatography
-
by using a Co2+-loaded metal-ion chelating TALON column
by using a TALON column
wild-type OxdRG: 104fold, recombinant OxdRG expressed in Escherichia coli JM109: 14.9fold
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL cells
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expression in Escherichia coli
overexpression in Escherichia coli
-
overexpression in Escherichia coli as his tagged fusion protein at the N-terminus
overexpression in Escherichia coli as His tagged fusion protein at the N-terminus, and as tagged fusion protein at the C-terminus
oxd gene, overexpression in Escherichia coli JM109, sequencing, gene cluster organisation
oxdA gene, overexpression in Escherichia coli BL21-Codon-Plus(DE3)-RIL, sequencing, gene cluster organisation
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E143A
-
the mutant shows about wild type enzyme activity
E143A/R178A
-
the mutant shows 29.4% activity compared to the wild type enzyme
H299A
-
mutant is unable to bind heme; the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme
H338A
-
the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme
N279A
-
the mutant shows 53.1% activity compared to the wild type enzyme
Q221A
-
the mutant shows about wild type enzyme activity
R178A
-
the mutant exhibits 19.1% of wild type activity and 1.62% of wild type heme content
S174A
-
the mutant shows about wild type enzyme activity
S219A
-
the mutant has completely lost activity despite unaffected heme content
S219C
-
the mutant shows 2.01% activity compared to the wild type enzyme
S219T
-
the mutant shows 26.1% activity compared to the wild type enzyme
Y234A
-
the mutant shows 170% activity compared to the wild type enzyme
E143A
-
the mutant shows about wild type enzyme activity
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H169A
-
the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme; the mutant shows 123% activity compared to the wild type enzyme
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H296A
-
the mutant shows 141% activity compared to the wild type enzyme
-
H299A
-
mutant is unable to bind heme; the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme
-
H320A
-
mutation does not affect the overall structure of OxdA but causes loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the Raman spectrum for the CO-bound form; the heme content and CD spectra in the far-UV region are almost identical to that of wild-type enzyme; the mutant shows 0.137% activity compared to the wild type enzyme
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S174A
-
the mutant shows about wild type enzyme activity
-
E143Q
loss in enzymatic activity, 14% of the wild-type
F306A
loss in enzymatic activity, 33% of the wild-type
H320A
loss in enzymatic activity, 11% of the wild-type
R178Q
loss in enzymatic activity, 36% of the wild-type
S219A
loss in enzymatic activity, 23% of the wild-type
H299A
-
the mutation leads to the loss of the ability to bind heme, thus becoming inactive
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
biotechnology
nitrile compounds are important intermediates in some industrial processes to produce nylon and acrylic fibers, insecticides, and pharmaceuticals. A more environmentally benign process of aldoxime dehydration is needed, for which a biological dehydration of aldoxime is a possible candidate
industry
synthesis