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Information on EC 4.2.1.84 - nitrile hydratase and Organism(s) Rhodococcus rhodochrous and UniProt Accession P29379

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EC Tree
     4 Lyases
         4.2 Carbon-oxygen lyases
             4.2.1 Hydro-lyases
                4.2.1.84 nitrile hydratase
IUBMB Comments
Acts on short-chain aliphatic nitriles, converting them into the corresponding amides. Does not act on these amides or on aromatic nitriles. cf. EC 3.5.5.1 nitrilase.
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Rhodococcus rhodochrous
UNIPROT: P29379 not found.
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Word Map
The taxonomic range for the selected organisms is: Rhodococcus rhodochrous
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
nhase, nitrile hydratase, nilco, l-nhase, h-nhase, co-type nhase, cobalt-containing nitrile hydratase, fe-nhase, ctnhase, mbnhase, more
SYNONYM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
3-cyanopyridine hydratase
-
-
-
-
acrylonitrile hydratase
-
-
-
-
aliphatic nitrile hydratase
-
-
-
-
H-NHase
H-nitrilase
-
-
-
-
high-molecular mass nitrile hydratase
-
-
high-molecular weight nitrile hydratase
-
hydratase, nitrile
-
-
-
-
L-Nhase
L-nitrilase
-
-
-
-
low-molecular mass nitrile hydratase
-
-
low-molecular weight nitrile hydratase
-
NHase
NI1 NHase
-
-
-
-
nitrilase
-
-
-
-
nitrile hydratase
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C-O bond cleavage by elimination of water
SYSTEMATIC NAME
IUBMB Comments
aliphatic-amide hydro-lyase (nitrile-forming)
Acts on short-chain aliphatic nitriles, converting them into the corresponding amides. Does not act on these amides or on aromatic nitriles. cf. EC 3.5.5.1 nitrilase.
CAS REGISTRY NUMBER
COMMENTARY hide
82391-37-5
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(R)-mandelonitrile + H2O
(R)-mandelamide
show the reaction diagram
wild-type enzyme catalyzes the conversion of rac-mandelonitrile to (S)-mandelamide with an enantiomeric excess of 52.6%
-
-
?
(S)-mandelonitrile + H2O
(S)-mandelamide
show the reaction diagram
wild-type enzyme catalyzes the conversion of rac-mandelonitrile to (S)-mandelamide with an enantiomeric excess of 52.6%
-
-
?
1-naphthylnitrile + H2O
1-naphthylamide
show the reaction diagram
-
-
-
-
?
2,2-dimethylcyclopropanecarbonitrile + H2O
2,2-dimethylcyclopropanecarboxamide
show the reaction diagram
-
-
-
-
?
2-cyanobenzamide + H2O
benzene-1,2-dicarboxamide
show the reaction diagram
-
-
-
?
2-cyanopyridine + H2O
pyridine-2-carbamide
show the reaction diagram
-
-
-
-
?
2-furonitrile + H2O
2-furoamide
show the reaction diagram
-
-
-
-
?
2-naphthylacetonitrile + H2O
2-naphthylacetamide
show the reaction diagram
-
-
-
-
?
3-cyanopyridine + H2O
nicotinamide
show the reaction diagram
3-cyanopyridine + H2O
pyridine-3-carbamide
show the reaction diagram
4-cyanobenzamide + H2O
benzene-1,4-dicarboxamide
show the reaction diagram
-
-
-
?
4-cyanopyridine + H2O
isonicotinamide
show the reaction diagram
-
-
-
-
?
5-cyanovaleramide
adiponitrile + H2O
show the reaction diagram
-
-
-
r
5-hydroxymethyl-2-furonitrile + H2O
5-hydroxymethyl-2-furamide
show the reaction diagram
-
-
-
-
?
acetonitrile + H2O
acetamide
show the reaction diagram
acrylamide + H2O
acrylonitrile
show the reaction diagram
-
-
-
-
?
acrylonitrile + H2O
2-propenoic acid amide
show the reaction diagram
acrylonitrile + H2O
acrylamide
show the reaction diagram
-
-
-
-
?
adiponitrile + 2 H2O
adipic acid amide
show the reaction diagram
wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide
-
-
?
adiponitrile + H2O
5-cyanovaleramide
show the reaction diagram
wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide
-
-
r
benzonitrile + H2O
benzamide
show the reaction diagram
-
-
-
-
?
benzonitrile + H2O
benzoic acid amide
show the reaction diagram
-
-
-
-
?
bromoxynil + H2O
?
show the reaction diagram
-
-
-
-
?
chloroacetonitrile + H2O
chloroacetamide
show the reaction diagram
-
-
-
-
?
chloroxynil acid + H2O
?
show the reaction diagram
-
-
-
-
?
chloroxynil amide + H2O
?
show the reaction diagram
-
-
-
-
?
crotononitrile + H2O
(E)-2-butenoic acid amide
show the reaction diagram
cyanoacetamide
malononitrile + H2O
show the reaction diagram
-
-
-
r
cyanopyrazine + H2O
pyrazincarbamide
show the reaction diagram
-
-
-
-
?
cyclopropylcyanide + H2O
?
show the reaction diagram
-
-
-
-
?
dichlobenil amide + H2O
?
show the reaction diagram
-
-
-
-
?
ethylene cyanhydrine + H2O
?
show the reaction diagram
-
-
-
-
?
ioxynil acid + H2O
?
show the reaction diagram
-
-
-
-
?
isobutyronitrile + H2O
isobutyramide
show the reaction diagram
-
-
-
-
?
malononitrile + 2 H2O
malonamide
show the reaction diagram
the use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. Mutant enzyme Y68T/W72Y produces 97.1% omega-cyanocarboxamide
-
-
?
malononitrile + H2O
cyanoacetamide
show the reaction diagram
the use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. Mutant enzyme Y68T/W72Y produces 97.1% omega-cyanocarboxamide
-
-
r
methacrylonitrile + H2O
methylacrylic acid amide
show the reaction diagram
-
-
-
-
?
n-butyronitrile + H2O
n-butyric acid amide
show the reaction diagram
-
-
-
-
?
phthalodinitrile + 2 H2O
phthalamide
show the reaction diagram
the wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme. Mutant enzyme Y68T/W72Y produces 100% 2-cyanobenzamide from phthalodinitrile
-
-
?
phthalodinitrile + H2O
2-cyanobenzamide
show the reaction diagram
the wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme. Mutant enzyme Y68T/W72Y produces 100% 2-cyanobenzamide from phthalodinitrile
-
-
?
propionitrile + H2O
propionic acid amide
show the reaction diagram
terephthalonitrile + 2 H2O
terephthalamide
show the reaction diagram
the wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. Mutant enzyme Y68T/W72Y produces 98.2% 4-cyanobenzamide from terephthalonitrile
-
-
?
terephthalonitrile + H2O
4-cyanobenzamide
show the reaction diagram
the wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. Mutant enzyme Y68T/W72Y produces 98.2% 4-cyanobenzamide from terephthalonitrile
-
-
?
trans-cinnamonitrile + H2O
trans-cinnamide
show the reaction diagram
-
-
-
-
?
additional information
?
-
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
(R)-mandelonitrile + H2O
(R)-mandelamide
show the reaction diagram
wild-type enzyme catalyzes the conversion of rac-mandelonitrile to (S)-mandelamide with an enantiomeric excess of 52.6%
-
-
?
(S)-mandelonitrile + H2O
(S)-mandelamide
show the reaction diagram
wild-type enzyme catalyzes the conversion of rac-mandelonitrile to (S)-mandelamide with an enantiomeric excess of 52.6%
-
-
?
additional information
?
-
-
NhhG forms a complex with the alpha-subunit of H-NHase. NhhAG is very similar to the mediator of L-NHase, NhlAE, which is a heterotrimer complex consisting of the cobalt-containing alpha-subunit of L-NHase and NhlE
-
-
?
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
CuSO4, CaCl2, ZnSO4, MnCl2, NaMnO4, FeCl3, FeSO4, AlCl3, BaCl2, SrCl2, NaWO4, SnCl2, BeSO4, NiCl2, PbCl2, LiCl and CoCl2 at 0.1% w/v neither inhibit nor enhance the enzyme activity
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
3-Cyanopyridine
1 M, 40% decrease of activity, low-molecular weight nitrile hydratase; high-molecular weight nitrile hydratase show almost no inhibition at 1 M
Ag+
-
0.01-1 mM AgNO3
Ag2SO4
-
total inhibition at 1 mM, L-NHase
AgNO3
-
complete inhibition at 1 mM
ammonium persulfate
-
4.5% inhibition at 1 mM
Ca2+
-
10% inhibition at 1 mM
Cd2+
-
8% inhibition at 1 mM
Co2+
-
2% inhibition at 1 mM
Cu2+
-
7% inhibition at 1 mM
diethyldithiocarbamate
-
0.1 mM
EDTA
-
12.1% inhibition at 1 mM
Fe2+
-
5% inhibition at 1 mM
H2O2
-
1 mM, no effect on L-NHase activity
Hg2+
-
HgCl2, 0.01-1 mM
hydroxylamine
iodoacetic acid
-
65% inhibition at 1 mM
L-ascorbic acid
-
54.6% inhibition at 1 mM
Mg2+
-
4% inhibition at 1 mM
Mn2+
-
8% inhibition at 1 mM
nicotinamide
0.5 M, sharp decrease in activity, low-molecular weight nitrile hydratase; 1.5 M, 40% decrease of activity, high-molecular weight nitrile hydratase
Pb2+
-
94.5% inhibition at 1 mM
phenyl hydrazine
-
68% inhibition at 1 mM
phenylhydrazine
-
1 mM
Semicarbazide
-
1 mM, weak inhibition
Sodium azide
-
26.6% inhibition at 1 mM
Urea
-
13.9% inhibition at 1 mM
additional information
-
not inhibited by dithiothreitol, phenylmethylsulfonylfluoride, and beta-mercaptoethanol
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
crotonamide
-
induces high and low molecular mass forms of the enzyme
cyclohexanecarboxamide
-
induces the low molecular mass form of the enzyme
dimethylformamide
-
addition to increase the accessibility of nitrile groups at concentration no higher than 0.5% is beneficial for activity
Urea
-
induces the high molecular mass form of the enzyme
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
43.8
(R)-mandelonitrile
pH 7.4, 10°C, wild-type enzyme
38.2 - 41.9
(S)-mandelonitrile
2.5
2-cyanobenzamide
pH 7.4, 35°C, wild-type enzyme
0.71 - 10
2-cyanopyrazine
0.56 - 21.7
2-Cyanopyridine
0.3 - 200
3-Cyanopyridine
2.3
4-cyanobenzamide
pH 7.4, 35°C, wild-type enzyme
0.28 - 18.5
4-cyanopyridine
13.2 - 127.6
5-cyanovaleramide
1.89 - 2.67
acrylonitrile
0.05 - 28.6
Benzonitrile
6.25 - 12.2
Chloroacetonitrile
4.55 - 4.88
Crotononitrile
20.7 - 88.2
cyanoacetamide
10
Cyanopyrazine
-
pH 7.0, 20ºC
1.51 - 1.85
Isobutyronitrile
0.44 - 6.76
Methacrylonitrile
0.65 - 21.7
n-butyronitrile
1.89 - 1.92
Propionitrile
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.74
(R)-mandelonitrile
pH 7.4, 10°C, wild-type enzyme
1.2 - 1.4
(S)-mandelonitrile
145.6
2-cyanobenzamide
pH 7.4, 35°C, wild-type enzyme
107.7
4-cyanobenzamide
pH 7.4, 35°C, wild-type enzyme
19.6 - 435.7
5-cyanovaleramide
39.1 - 417.2
cyanoacetamide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0168
(R)-mandelonitrile
pH 7.4, 10°C, wild-type enzyme
0.032 - 0.034
(S)-mandelonitrile
58.2
2-cyanobenzamide
pH 7.4, 35°C, wild-type enzyme
46.8
4-cyanobenzamide
pH 7.4, 35°C, wild-type enzyme
0.2 - 33
5-cyanovaleramide
0.4 - 20.2
cyanoacetamide
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
2.4
-
purified recombinant wild-type apo-H-NHase, pH 7.5, 20°C
2.8
-
purified recombinant V5L mutant apo-H-NHase, pH 7.5, 20°C
270
-
purified recombinant V5L mutant R-H-NHase, pH 7.5, 20°C
275
-
purified recombinant wild-type R-H-NHase, pH 7.5, 20°C
371
-
purified enzyme
4.16
-
cell-free extract, at pH 8.0, at 40°C
46.83
-
after 11.3fold purification, at pH 8.0, at 40°C
5.21
-
with the addition of 0.1% w/v CoCl2
additional information
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 8
-
about 50% of activity maximum at pH 5, about 60% of activity maximum at pH 8
6 - 8.5
-
H-NHase
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20
-
H-NHase activity assay at
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
incorporation of cobalt into L-NHase in a mode of post-translational maturation, i.e. self-subunit swapping. NhlE is recognized as a self-subunit swapping chaperone, mechanism, detailed overview
UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION?
NHB1_RHORH
229
0
26322
Swiss-Prot
-
NHB2_RHORH
226
0
25201
Swiss-Prot
-
NHA1_RHORH
203
0
22835
Swiss-Prot
-
NHA2_RHORH
207
0
22848
Swiss-Prot
-
Q692F3_RHORH
203
0
22791
TrEMBL
-
A0A6I6YKY8_RHORH
207
0
22848
TrEMBL
-
A0A6I6Y6E2_RHORH
226
0
25201
TrEMBL
-
Q692F4_RHORH
229
0
26294
TrEMBL
-
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
101000
-
sedimentation equilibrium, low molecular mass enzyme, L-NHase
130000
-
gel filtration, low molecular mass enzyme, L-NHase
25040
-
1 * 25040 + 1 * 30600, estimated from SDS-PAGE
26000
29000
30600
-
1 * 25040 + 1 * 30600, estimated from SDS-PAGE
505000 - 530000
520000
-
high molecular mass enzyme, H-NHase
86000
-
holoenzyme, native PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
heterodimer
-
1 * 25040 + 1 * 30600, estimated from SDS-PAGE
oligomer
alpha10beta10, 10 * 22800 (alpha) + 10 * 26300 (beta), calculated from sequence
tetramer
additional information
-
NhhG forms a complex with the alpha-subunit of H-NHase. NhhAG is very similar to the mediator of L-NHase, NhlAE, which is a heterotrimer complex consisting of the cobaltcontaining alpha-subunit of L-NHase and NhlE. Formation of large-sized complexes during self-subunit swapping in H-NHase. Self-subunit swapping mechanisms, detailed overview
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A51L
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 74.3%
alphaV5L
-
site-directed mutagenesis, exchange in the H-NHase does not influence the catalytic activity or the Co2+ content
F37H
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 96.8%
F37H/F51L
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 94.6%
F37H/L48A
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 95.1%
F37Y
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 88.7%
F51Q
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 60.2%
L48Q
wild-type enzyme catalyzes the conversion of (S)-mandelamide with an enantiomeric excess of 52.6%, the beta-subunit mutant enzyme catalyzes the reaction with an enantiomeric excess of 59.7%
W72Y
with adiponitrile as substrate the wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. The use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. The wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. The wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme
Y68T
with adiponitrile as substrate the wild-type enzyme forms only adipamide after a 4 h reaction. Y68T and W72Y mutations cause a significant shift in product formation and form primarily 5-cyanovaleramide. The use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Variants Y68T and W72Y show a drastic change in regiospecificity by producing mainly the omega-cyanocarboxamide, cyanoacetamide, at a relatively low malononitrile conversion. The wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2%. Variants Y68T and W72Y show a change in regiospecificity by producing mainly the 4-cyanobenzamide. The wild-type enzyme forms 100% phthalamide from phthalodinitrile. The mutant enzymes Y68T and W72Y result in a higher 2-cyanobenzamide formation than their parent enzyme
Y68T/W72Y
the mutant enzyme displays a totally different regioselectivity towards dinitriles than its parent enzyme. The use of the wild-type enzyme leads to malonamide formation with 97.3% malononitrile conversion. Mutant enzyme Y68T/W72Y produces 97.1% omega-cyanocarboxamide. The wild-type enzyme forms only adipamide after a 4 h reaction. Mutant enzyme Y68T/W72Y produces 100% 5-cyanovaleramide. The wild-type enzyme prefers terephthalonitrile to catalyze mainly into terephthalamide (84.3%) with a conversion up to 99.2 %. Mutant enzyme Y68T/W72Y produces 98.2% 4-cyanobenzamide from terephthalonitrile. The wild-type enzyme forms 100% phthalamide from phthalodinitrile. Mutant enzyme Y68T/W72Y produces 100% 2-cyanobenzamide from phthalodinitrile
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5 - 10
-
the pH below 5 and above 10 drastically decreases the activity of purified NHase
703995
6 - 9
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40 - 50
-
half-life is 2 h at 40°C and 0.5 h at 50°C, below and above 40°C NHase activity decreases drastically
additional information
-
the enzyme is heat-stable
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
organic acids stabilize, stable for more than 1 month in 0.1 M HEPES/KOH, pH 7.2, with 44 mM n-butyric acid, n-valeric acid, isovaleric acid, isobutyric acid or n-caproic acid
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimethylformamide
-
30% v/v, no residual activity
STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 2 weeks, 90% residual activity, with addition of 8% glycerol, complete loss of activity within one week
-
organic acids stabilize, stable for more than 1 month in 0.1 M HEPES/KOH, pH 7.2, with 44 mM n-butyric acid, n-valeric acid, isovaleric acid, isobutyric acid or n-caproic acid
-
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
ammonium sulfate fractionation, chromatography on DEAE-Sephacel, octyl-Sepharose CL-4B and phenyl-Sepharose CL-4B, 4.91fold purification
-
ammonium sulfate fractionation, chromatography on DEAE-Sephacel, phenyl-Sepharose and Sephacryl S-300, 3.31fold purification
-
ammonium sulfate fractionation, chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B and gel filtration
-
ammonium sulfate fractionation, Sephacryl S 300 gel filtration, and DEAE column chromatography
-
recombinant L-NHase, NhlAE, and the NhhA-NhlE hybrid mediator complex from Rhodococcus rhodochrous strain ATCC12674 and Rhodococcus fascians DSM43985 by ammonium sulfate fractionation anion exchange chromatography, dialysis, gel filtration, and another step of anion exchange chromatography
-
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli
high molecular mass-NHase and low molecular mass-NHase genes cloned into Escherichia coli
-
overexpressed in Escherichia coli by codon-optimization, engineering of Ribosome Binding Site and spacer sequences
separate expression of L-NHase, NhlAE, an the NhhA-NhlE hybrid mediator complex in Rhodococcus rhodochrous strain ATCC12674 and Rhodococcus fascians DSM43985. Necessity of gene nhhG for functional H-NHase expression
-
the nhhBAG gene of Rhodococcus rhodochrous M33 that encodes nitrile hydratase is cloned and expressed in Corynebacterium glutamicum under the control of an ilvC promoter. To overexpress the nitrile hydratase, five types of plasmid variants are constructed by introducing mutations into 80 nucleotides near the translational initiation region of nhhB. Of them, pNBM4 with seven mutations shows the highest NHase activity, exhibiting higher expression levels of NhhB and NhhA than wild-type pNBW33, mainly owing to decreased secondary-structure stability and an introduction of a conserved Shine-Dalgarno sequence in the translational initiation region. In a fed-batch culture of recombinant Corynebacterium cells harboring pNBM4, the cell density reaches 53.4 g dry cell weight/l within 18 h, and the specific and total enzyme activities are estimated to be 0.0373 mM/min/mg dry cell weight and 1.992 mM/min/ml, respectively
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
industry
biotransformation of nitrile. Nitrile hydratase from Rhodococcus rhodochrous J1 is used for industrial production of acrylamide and nicotinamide. Production of enzyme by recombinant Escherichia coli is superior to that in R. rhodochrous J1. Genetically engineered Escherichia coli can be used for industrial applications instead of Rhodococcus rhodochrous J1. High-molecular weight nitrile hydratase may be more suitable for industrial application than low-molecular weight nitrile hydratase because of its higher product tolerance, which would lead to a high product concentration
synthesis
additional information
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Rhodococcus rhodochrous PA-34 converts benzonitrile herbicides into amides, but the strain does not hydrolyze 2,6-dichlorobenzamide into 2,6-dichlorobenzoic acid. Transformation of nitriles into amides decreases acute toxicities for chloroxynil and dichlobenil, but increases them for bromoxynil and ioxynil. The amides inhibit root growth in Lactuca sativa less than the nitriles but more than the acids. The conversion of the nitrile group may be the first step in the mineralization of benzonitrile herbicides but cannot be itself considered to be a detoxification
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Kobayashi, M.; Nishiyama, M.; Nagasawa, T.; Horinouchi, S.; Beppu, T.; Yamada, H.
Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1
Biochim. Biophys. Acta
1129
23-33
1991
Rhodococcus rhodochrous, Rhodococcus rhodochrous J1, Rhodococcus sp., Rhodococcus sp. N-774
Manually annotated by BRENDA team
Nagasawa, T.; Takeuchi, K.; Yamada, H.
Occurrence of a cobalt-induced and cobalt-containing nitrile hydratase in Rhodococcus rhodochrous J1
Biochem. Biophys. Res. Commun.
155
1008-1016
1988
Pseudomonas chlororaphis, Rhodococcus rhodochrous, Rhodococcus rhodochrous J1
Manually annotated by BRENDA team
Duran, R.; Chion, C.K.N.C.K.; Bigey, F.; Arnaud, A.; Galzy, P.
The N-terminal amino acid sequences of Brevibacterium sp. R312 nitrile hydratase
J. Basic Microbiol.
32
13-19
1992
Arthrobacter sp., Brevibacterium sp., Brevibacterium sp. R312, Corynebacterium sp., Pseudomonas chlororaphis, Rhodococcus rhodochrous, Rhodococcus rhodochrous J1, Rhodococcus sp., Rhodococcus sp. 7, Rhodococcus sp. N-774
Manually annotated by BRENDA team
Nagasawa, T.; Takeuchi, K.; Yamada, H.
Characterization of a new cobalt-containing nitrile hydratase purified from urea-induced cells of Rhodococcus rhodochrous J1
Eur. J. Biochem.
196
581-589
1991
Rhodococcus rhodochrous, Rhodococcus rhodochrous J1
Manually annotated by BRENDA team
Yamada, H.; Kobayashi, M.
Nitrile hydratase and its application to industrial production of acrylamide
Biosci. Biotechnol. Biochem.
60
1391-1400
1996
Agrobacterium tumefaciens, Albifimbria verrucaria, Arthrobacter sp., Pseudomonas chlororaphis, Pseudomonas chlororaphis B23, Rhodococcus rhodochrous, Rhodococcus rhodochrous J1, Rhodococcus sp.
Manually annotated by BRENDA team
Kaufmann, G.; Dautzenberg, H.; Henkel, H.; Mller, G.; Schfer, T.; Undeutsch, B.; Oettel, M.
Nitrile hydratase from Rhodococcus erythropolis: metabolization of steroidal compounds with a nitrile group
Steroids
64
535-540
1999
Rhodococcus erythropolis, Rhodococcus rhodochrous
Manually annotated by BRENDA team
Wieser, M.; Takeuchi, K.; Wada, Y.; Yamada, H.; Nagasawa, T.
Low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1: purification, substrate specificity and comparison with the analogous high-molecular-mass enzyme
FEMS Microbiol. Lett.
169
17-22
1998
Rhodococcus rhodochrous, Rhodococcus rhodochrous J1
-
Manually annotated by BRENDA team
Kobayashi, M.; Shimizu, S.
Metalloenzyme nitrile hydratase: structure, regulation, and application to biotechnology
Nat. Biotechnol.
16
733-736
1998
Pseudomonas chlororaphis, Pseudomonas chlororaphis B23, Pseudomonas putida, Rhodococcus rhodochrous, Rhodococcus rhodochrous J1, Rhodococcus sp., Rhodococcus sp. N-774, Rhodococcus sp. R312
Manually annotated by BRENDA team
Tauber, M.M.; Cavaco-Paulo, A.; Robra, K.; Gubitz, G.M.
Nitrile hydratase and amidase from Rhodococcus rhodochrous hydrolyze acrylic fibers and granular polyacrylonitriles
Appl. Environ. Microbiol.
66
1634-1638
2000
Rhodococcus rhodochrous, Rhodococcus rhodochrous NCIMB 11216
Manually annotated by BRENDA team
Petersen, M.; Kiener, A.
Biocatalysis - Preparation and functionalization of N-heterocycles
Green Chem.
4
99-106
1999
Rhodococcus rhodochrous, Rhodococcus rhodochrous J1
-
Manually annotated by BRENDA team
Kashiwagi, M.; Fuhshuku, K.I.; Sugai, T.
Control of the nitrile-hydrolyzing enzyme activity in Rhodococcus rhodochrous IFO 15564: preferential action of nitrile hydratase and amidase depending on the reaction condition factors and its application to the one-pot preparation of amides from aldehydes
J. Mol. Catal. B
29
249-258
2004
Rhodococcus rhodochrous, Rhodococcus rhodochrous IFO 15564
-
Manually annotated by BRENDA team
Yeom, S.; Kim, H.; Oh, D.
Enantioselective production of 2,2-dimethylcyclopropane carboxylic acid from 2,2-dimethylcyclopropane carbonitrile using the nitrile hydratase and amidase of Rhodococcus erythropolis ATCC 25544
Enzyme Microb. Technol.
41
842-848
2007
Rhodococcus erythropolis, Rhodococcus rhodochrous, Nocardia transvalensis, Comamonas oleophilus
-
Manually annotated by BRENDA team
Prasad, S.; Raj, J.; Bhalla, T.
Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34
Indian J. Microbiol.
49
237-242
2009
Rhodococcus rhodochrous, Rhodococcus rhodochrous PA-34
Manually annotated by BRENDA team
Zhou, Z.; Hashimoto, Y.; Cui, T.; Washizawa, Y.; Mino, H.; Kobayashi, M.
Unique biogenesis of high-molecular mass multimeric metalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swapping maturation
Biochemistry
49
9638-9648
2010
Rhodococcus rhodochrous, Rhodococcus rhodochrous J1
Manually annotated by BRENDA team
Vesela, A.B.; Pelantova, H.; Sulc, M.; Mackova, M.; Lovecka, P.; Thimova, M.; Pasquarelli, F.; Picmanova, M.; Patek, M.; Bhalla, T.C.; Martinkova, L.
Biotransformation of benzonitrile herbicides via the nitrile hydratase-amidase pathway in Rhodococci
J. Ind. Microbiol. Biotechnol.
39
1811-1819
2012
Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus erythropolis A4, Rhodococcus rhodochrous PA-34
Manually annotated by BRENDA team
Kang, M.S.; Han, S.S.; Kim, M.Y.; Kim, B.Y.; Huh, J.P.; Kim, H.S.; Lee, J.H.
High-level expression in Corynebacterium glutamicum of nitrile hydratase from Rhodococcus rhodochrous for acrylamide production
Appl. Microbiol. Biotechnol.
98
4379-4387
2014
Rhodococcus rhodochrous, Rhodococcus rhodochrous M33
Manually annotated by BRENDA team
Cheng, Z.; Peplowski, L.; Cui, W.; Xia, Y.; Liu, Z.; Zhang, J.; Kobayashi, M.; Zhou, Z.
Identification of key residues modulating the stereoselectivity of nitrile hydratase toward rac-mandelonitrile by semi-rational engineering
Biotechnol. Bioeng.
115
524-535
2018
Rhodococcus rhodochrous (P29378 AND P29379), Rhodococcus rhodochrous J1 (P29378 AND P29379)
Manually annotated by BRENDA team
Cheng, Z.; Cui, W.; Xia, Y.; Peplowski, L.; Kobayashi, M.; Zhou, Z.
Modulation of nitrile hydratase regioselectivity towards dinitriles by tailoring the substrate binding pocket residues
ChemCatChem
10
449-458
2018
Rhodococcus rhodochrous (P29378 AND P29379), Rhodococcus rhodochrous J1 (P29378 AND P29379)
-
Manually annotated by BRENDA team
Lan, Y.; Zhang, X.; Liu, Z.; Zhou, L.; Shen, R.; Zhong, X.; Cui, W.; Zhou, Z.
Overexpression and characterization of two types of nitrile hydratases from Rhodococcus rhodochrous J1
PLoS ONE
12
e0179833
2017
Rhodococcus rhodochrous (P21219 AND P21220), Rhodococcus rhodochrous (P29378 AND P29379), Rhodococcus rhodochrous, Rhodococcus rhodochrous J1 (P21219 AND P21220), Rhodococcus rhodochrous J1 (P29378 AND P29379), Rhodococcus rhodochrous J1
Manually annotated by BRENDA team