Information on EC 4.2.1.103 - cyclohexyl-isocyanide hydratase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
4.2.1.103
-
RECOMMENDED NAME
GeneOntology No.
cyclohexyl-isocyanide hydratase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N-cyclohexylformamide = cyclohexyl isocyanide + H2O
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
addition
-
-
-
-
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Caprolactam degradation
-
-
Microbial metabolism in diverse environments
-
-
SYSTEMATIC NAME
IUBMB Comments
N-cyclohexylformamide hydro-lyase (cyclohexyl-isocyanide-forming)
The enzyme from Pseudomonas putida strain N19-2 can also catalyse the hydration of other isonitriles to the corresponding N-substituted formamides. The enzyme has no metal requirements.
CAS REGISTRY NUMBER
COMMENTARY hide
358974-06-8
-
ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
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
1-pentyl isocyanide + H2O
N-1-pentylformamide
show the reaction diagram
2-naphthyl isocyanide + H2O
N-(2-naphthyl)formamide
show the reaction diagram
benzyl isocyanide
N-phenylformamide
show the reaction diagram
benzyl isocyanide + H2O
N-benzylformamide
show the reaction diagram
cyclohexyl isocyanide + H2O
N-cyclohexyl formamide
show the reaction diagram
cyclohexyl isocyanide + H2O
N-cyclohexylformamide
show the reaction diagram
ethyl isocyanoacetate + H2O
N-formylglycine ethyl ester
show the reaction diagram
-
-
-
?
isocyanomethyl phosphonic acid diethyl ether + H2O
formylaminomethyl-phosphonic acid diethyl ester
show the reaction diagram
methyl isocyanoacetate + H2O
N-formylglycine methyl ester
show the reaction diagram
-
-
-
?
tert-butyl isocyanide + H2O
N-tert-butylformamide
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
cyclohexyl isocyanide + H2O
N-cyclohexyl formamide
show the reaction diagram
cyclohexyl isocyanide + H2O
N-cyclohexylformamide
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
no metal requirements
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4-chloromercuribenzoate
99.8% inhibition at 1 mM
5,5'-dithiobis-(2-nitrobenzoate)
34.9% inhibition at 1 mM
8-hydroxyquinoline
26.1% inhibition at 1 mM
Ag+
complete inhibition at 1 mM
AgNO3
-
1 mM, 99% inhibition
Al3+
21.4% inhibition at 1 mM
ammonium persulfate
-
1 mM, 23% inhibition
Cd2+
71.6% inhibition at 1 mM
CdCl2
-
1 mM, 87% inhibition
Co2+
89.5% inhibition at 1 mM
CoCl2
-
1 mM, 98% inhibition
Cu2+
complete inhibition at 1 mM
CuSO4
-
1 mM, 98% inhibition
Diethyldicarbamate
-
1 mM, 20% inhibition
-
Fe2+
7.6% inhibition at 1 mM
Fe3+
19.7% inhibition at 1 mM
FeSO4
-
1 mM, 50% inhibition
Hg2+
complete inhibition at 1 mM
HgCl2
-
1 mM, 99% inhibition
hydroxylamine
-
1 mM, 10% inhibition
iodoacetate
-
1 mM, 97% inhibition
KCN
-
1 mM, 26% inhibition
N-ethylmaleimide
48.1% inhibition at 1 mM
NEM
-
1 mM,51% inhibition
Ni2+
95.0% inhibition at 1 mM
NiSO4
-
1 mM, 98% inhibition
Pb2+
11.9% inhibition at 1 mM
PCMB
-
1 mM, 50% inhibition
phenylmethanesulfonyl fluoride
-
1 mM, 57% inhibition
PMSF
49.4% inhibition at 1 mM
Sn2+
41.7% inhibition at 1 mM
SnCl2
-
1 mM, 16% inhibition
Zn2+
51.9% inhibition at 1 mM
ZnCl2
-
1 mM, 34% inhibition
additional information
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0203 - 0.0522
2-naphthyl isocyanide
0.95 - 16.2
cyclohexyl isocyanide
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.092 - 0.234
2-naphthyl isocyanide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
4.15 - 7.62
2-naphthyl isocyanide
12173
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.935
purified enzyme, substrate is cyclohexyl isocyanide, pH 7.5, 25C
1.4
purified recombinant mutant T102A
15.4
purified recombinant mutant E81Q
17.3
purified recombinant wild-type enzyme
18.7
purified recombinant mutant E79Q
additional information
-
-
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9
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pH 5.5: about 45% of maximal activity, potassium phosphate buffer, pH 9.0: about 30% of maximal activity, NH4OH/NH4Cl buffer
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 45
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20C: about 55% of maximal activity, 45C: about 55% of maximal activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
Pseudomonas fluorescens (strain ATCC BAA-477 / NRRL B-23932 / Pf-5)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
24000
2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE
24158
2 * 24158, N-terminally GSH-tagged enzyme, sequence calculation, 2 * 24000, SDS-PAGE
24211
x * 24211, amino acid sequence calculation
43000
12 * 43000, SDS-PAGE
43700
recombinant ICH, sedimentation equilibrium ultracentrifugation
59000
-
2 * 59000, SDS-PAGE
530000
gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
homodimer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified recombinant wild-type and several site-directed mutants, hanging drop vapor diffusion method, for wild-type, C101S, and C101A enzymes: 0.002 ml of 20 mg/ml protein solution is mixed with 0.002 ml of reservoir solution containing 24-26% PEG 3350, 200-250 mM magnesium chloride, 100 mM CHES, pH 9.3, or 100 mM Tris-HCl, pH 8.6, and equilibration against 0.5 ml of reservoir solution, 1-3 days, for D17E enzyme: 0.002 ml of 24 mg/ml protein is mixed with 0.002 ml of reservoir solution containing 12% PEG 4000, 240 mM ammonium acetate, 100 mM sodium acetate, pH 4.6, 2-3 days at room temperature, for T102S enzyme: 0.002 ml of 17 mg/ml protein solution is mixed with 0.002 ml of reservoir solution containing 19-22% PEG 4000, 140-160 mM sodium citrate, pH 5.6, 2-3 days at room temperature, X-ray diffraction structure determination and analysis at 1.0-1.9 A resolution
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.5 - 9.7
most stable in the range of pH 5.5-9.7
715552
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10
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30 min, 10% v/v glycerol, 3% loss of activity
20
-
30 min, 10% v/v glycerol, 11% loss of activity
30
-
30 min, 10% v/v glycerol, 16% loss of activity
35 - 40
most stable at 35C, enzyme activity is rapidly lost above 40C
35
-
30 min, 10% v/v glycerol, 22% loss of activity
40
-
30 min, 10% v/v glycerol, 31% loss of activity
45
-
30 min, 10% v/v glycerol, 47% loss of activity
50
-
30 min, 10% v/v glycerol, 67% loss of activity
55
-
30 min, 10% v/v glycerol, 81% loss of activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
native enzyme 14.6fold by ammonium sulfate fractionation followed by two steps of hydrophobic interaction chromatography
recombinant N-terminally His-tagged wild-type and mutant ICHs from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, cleavage of the His-tag through thrombin, and benzamidine affinity chromatography. The final recombinant ICH protein has three vector-derived amino acids at the N terminus, GSH
recombinant wild-type and mutants from Escherichia coli
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression of N-terminally His-tagged wild-type and mutant ICHs in Escherichia coli strain BL21(DE3)
gene inhA, DNA and amino acid sequence determination and analysis, overexpression of wild-type and mutant enzyme in Escherichia coli
InhB, DNA and amino acid sequence determination and analysis, cloning and expression in Escherichia coli strain DH10B
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
the enzyme is induced by N-benzylformamide and isonitriles benzyl isocyanide and cyclohexyl isocyanide, while benzylamine and N-benzylacetamide do not induce isonitrile hydratase activity
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C101A
site-directed mutagenesis, inactive mutant
C101S
site-directed mutagenesis, inactive mutant, the mutant shows a stronger electron density for a water molecule between Asp17 and residue 101 compared to the wild-type enzyme
D17E
site-directed mutagenesis, the mutant is almost inactive
D17N
site-directed mutagenesis, the mutant is almost inactive
D17V
site-directed mutagenesis, the mutant is almost inactive
T102S
site-directed mutagenesis, the mutant shows about 2fold increased activity compared to the wild-type enzyme
T102V
site-directed mutagenesis, the mutant shows slightly increased activity compared to the wild-type enzyme, the mutation results in a substrate-inhibited enzyme
C101A
-
site-directed mutagenesis, inactive mutant
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C101S
-
site-directed mutagenesis, inactive mutant, the mutant shows a stronger electron density for a water molecule between Asp17 and residue 101 compared to the wild-type enzyme
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D17E
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site-directed mutagenesis, the mutant is almost inactive
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D17N
-
site-directed mutagenesis, the mutant is almost inactive
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D17V
-
site-directed mutagenesis, the mutant is almost inactive
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C101A
site-directed mutagenesis, inactive mutant
E79Q
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
E81Q
site-directed mutagenesis, slightly decreased activity compared to the wild-type enzyme
T102A
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
C101A
-
site-directed mutagenesis, inactive mutant
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E79Q
-
site-directed mutagenesis, slightly increased activity compared to the wild-type enzyme
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E81Q
-
site-directed mutagenesis, slightly decreased activity compared to the wild-type enzyme
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T102A
-
site-directed mutagenesis, highly reduced activity compared to the wild-type enzyme
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Show AA Sequence (186 entries)
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