Information on EC 3.5.1.49 - formamidase

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

EC NUMBER
COMMENTARY hide
3.5.1.49
-
RECOMMENDED NAME
GeneOntology No.
formamidase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
formamide + H2O = formate + NH3
show the reaction diagram
Also acts, more slowly, on acetamide, propanamide and butanamide
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
carboxylic acid amide hydrolysis
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
cyanide detoxification II
-
-
Cyanoamino acid metabolism
-
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Glyoxylate and dicarboxylate metabolism
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Nitrogen metabolism
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SYSTEMATIC NAME
IUBMB Comments
formamide amidohydrolase
Also acts, more slowly, on acetamide, propanamide and butanamide.
CAS REGISTRY NUMBER
COMMENTARY hide
9013-59-6
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SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetamide + H2O
acetate + NH3
show the reaction diagram
acetamide + H2O
acetic acid + NH3
show the reaction diagram
acrylamide + H2O
acrylic acid + NH3
show the reaction diagram
-
very low activity
-
?
butyramide + H2O
butyric acid + NH3
show the reaction diagram
formamide + H2O
formate + NH3
show the reaction diagram
propionamide + H2O
propionate + NH3
show the reaction diagram
-
-
-
?
propionamide + H2O
propionic acid + NH3
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
acetamide + H2O
acetate + NH3
show the reaction diagram
formamide + H2O
formate + NH3
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
-
the presence of K+, Na+, Ca2+, Mg2+, and Pb2+ has no significant effect on enzyme activity
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Hg2+
-
complete inhibition at concentrations above 15 mM
iodoacetamide
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complete inhibition at concentrations above 15 mM
iodoacetate
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2.5 mM, 55% inhibition
Thiourea
-
competitive inhibition, Ki: 4.6 mM
additional information
-
the presence of EDTA and dithiothreitol has no significant effect on enzyme activity
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
2.1 - 108
Formamide
120
Propionamide
recombinant formamidase, pH and temperature not specified in the publication
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
60.7
Formamide
Methylophilus methylotrophus
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-
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.05
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amidase activity wild-type Helicobacter pylori 26695, pH 7.0, 0 micromol NiCl2 supplementation; amidase activity wild-type Helicobacter pylori 26695, pH 7.0, 1 micromol NiCl2 supplementation
0.1
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amidase activity wild-type Helicobacter pylori 26695, pH 7.0; amidase activity wild-type Helicobacter pylori 26695, pH 7.0, 100 micromol NiCl2 supplementation
0.5
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amidase activity wild-type Helicobacter pylori 26695, pH 7.0, 500 micromol NiCl2 supplementation
0.55
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amidase activity wild-type Helicobacter pylori 26695, pH 7.0, 1000 micromol NiCl2 supplementation
0.6
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amidase activity wild-type Helicobacter pylori 26695, pH 5.5
2.73
purified recombinant formamidase
5
-
formamidase activity wild-type Helicobacter pylori 26695, pH 7.0; urease activity wild-type Helicobacter pylori 26695, pH 7.0
5.2
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formamidase activity wild-type Helicobacter pylori 26695, pH 7.0, 1000 micromol NiCl2 supplementation
6
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formamidase activity wild-type Helicobacter pylori 26695, pH 7.0, 0 micromol NiCl2 supplementation; formamidase activity wild-type Helicobacter pylori 26695, pH 7.0, 1 micromol NiCl2 supplementation
8
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formamidase activity wild-type Helicobacter pylori 26695, pH 7.0, 500 micromol NiCl2 supplementation; urease activity wild-type Helicobacter pylori 26695, pH 7.0, 0 micromol NiCl2 supplementation
8.1
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formamidase activity wild-type Helicobacter pylori 26695, pH 7.0, 100 micromol NiCl2 supplementation
20
-
urease activity wild-type Helicobacter pylori 26695, pH 5.5
25
-
formamidase activity wild-type Helicobacter pylori 26695, pH 5.5
30
-
urease activity wild-type Helicobacter pylori 26695, pH 7.0, 1 micromol NiCl2 supplementation
60
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urease activity wild-type Helicobacter pylori 26695, pH 7.0, 100 micromol NiCl2 supplementation
70
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urease activity wild-type Helicobacter pylori 26695, pH 7.0, 1000 micromol NiCl2 supplementation
75
-
urease activity wild-type Helicobacter pylori 26695, pH 7.0, 500 micromol NiCl2 supplementation
1040
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pH 7.4, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
40
recombinant formamidase
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30 - 50
recombinant formamidase
35 - 70
-
about 50% activity at 35 and 70°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Bacillus cereus (strain ATCC 14579 / DSM 31 / JCM 2152 / NBRC 15305 / NCIMB 9373 / NRRL B-3711)
Bacillus cereus (strain ATCC 14579 / DSM 31 / JCM 2152 / NBRC 15305 / NCIMB 9373 / NRRL B-3711)
Delftia acidovorans (strain DSM 14801 / SPH-1)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
Helicobacter pylori (strain ATCC 700392 / 26695)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
32000
-
4 * 32000, calculated from amino acid sequence
37300
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4 * 3400, SDS-PAGE, 4 * 37300, deduced from gene sequence
38000
-
4 * 38000, His-tagged enzyme, SDS-PAGE
38632
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4 * 38632, His-tagged enzyme, calculated from amino acid sequence
43300
4 * 43300, native state, native PAGE
44481
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3 * 44481, electrospray mass spectrometry
49770
calculated from amino acid sequence
51000
-
alpha2, 2 * 51000, SDS-PAGE
52000
recombinant His-tagged enzyme
123000
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gel filtration
133000 - 137000
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gel filtration, non-denaturing PAGE
135000 - 160000
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gel filtration
160000
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gel filtration
180000
SDS-PAGE, the 180000 Da purified protein yields a heat-denatured species of 45000 Da
191000
purified formamidase, native PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dimer
-
alpha2, 2 * 51000, SDS-PAGE
homotetramer
trimer
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3 * 44481, electrospray mass spectrometry
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
hanging drop vapor diffusion method, using 0.2 M calcium acetate, 0.1 M sodium cacodylate and 18% (w/v) PEG 8000 pH 6.2 for the native protein or 18% (w/v) PEG 8000, 0.2 M calcium acetate and 0.1 M sodium acetate pH 5.8 for the SeMet-substituted protein
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crystal structure of AmiF is solved to 1.75 A resolution using single-wavelength anomalous dispersion methods. The structure consists of a homohexamer related by 3fold symmetry in which each subunit has an alpha-beta-beta-alpha four-layer architecture characteristic of the nitrilase superfamily
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50 - 75
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after incubation at the optimal temperature for activity of the enzyme (50°C) for 40 h, the enzyme maintains around 95% of its initial activity. Activity is gradually lost when the enzyme is incubated at temperatures over 65°C for 30 min. The melting temperature is at 73.5°C
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in the soluble form by Escherichia coli XL1 Blue MRF cells (5mg fusion protein per liter culture), purified on a Glutathione Sepharose 4B Resin column, cleaved with thrombin
glutatione Sepharose column chromatography, DEAE Sepharose column chromatography, and phenyl Sepharose column chromatography
MagneHis protein purification system
Ni-NTA His-binding affinity chromatography, gel filtration on a Sephacryl S-200 column
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recombinant protein
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Talon metal-affinity resin column chromatography and Superdex 200 gel filtration
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expression in Escherichia coli
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Escherichia coli strain Rosetta (DE3), 20-residue His-tag at N-terminus
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expressed in Escherichia coli BL21 Star (DE3) cells
expressed in Escherichia coli BL21(DE3) cells
expression in Escherichia coli
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into the SalI/NotI sites of the Vector pGEX-4T-3, fused to glutathione S-transferase (GST)
the native soluble protein is expressed in Escherichia coli Rosetta (DE3) cells, the SeMet-substituted protein is expressed in the methionine-auxotrophic Escherichia coli strain B834 (DE3)
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
E140D
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the mutation does not significantly alter enzyme folding and shows no detectable activity
W136H
-
this mutation causes the absence of overexpression of the enzyme
Y191F
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the mutation does not significantly alter enzyme folding and shows 11.8% activity compared to the wild type enzyme
E140D
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the mutation does not significantly alter enzyme folding and shows no detectable activity
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W136H
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this mutation causes the absence of overexpression of the enzyme
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Y191F
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the mutation does not significantly alter enzyme folding and shows 11.8% activity compared to the wild type enzyme
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SeMet-substituted form
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MAD data collection
C166A
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no enzymic activity
C166S
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no enzymic activity
fur mutant
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amidase activity at pH 5.5: wild-type 0.6, mutant 3.0 - at pH 7.0: wild-type 0.1, mutant 2.9; formamidase activity at pH 5.5: wild-type 25, mutant 41 - at pH 7.0: wild-type 5, mutant 41; urease activity at pH 5.5: wild-type 20, mutant 23 - at pH 7.0: wild-type 4, mutant 3
nikR fur double mutant
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amidase activity at pH 5.5: wild-type 0.6, mutant 1.9 - at pH 7.0: wild-type 0.1, mutant 3.0; formamidase activity at pH 5.5: wild-type 25, mutant 30 - at pH 7.0: wild-type 5, mutant 40; urease activity at pH 5.5: wild-type 20, mutant 14 - at pH 7.0: wild-type 4, mutant 6
nikR mutant
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amidase activity at pH 5.5: wild-type 0.6, mutant 0.2 - at pH 7.0: wild-type 0.1, mutant 0.1; formamidase activity at pH 5.5: wild-type 25, mutant 8 - at pH 7.0: wild-type 5, mutant 5; urease activity at pH 5.5: wild-type 20, mutant 13 - at pH 7.0: wild-type 4, mutant 5
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