Information on EC 3.5.4.42 - N-isopropylammelide isopropylaminohydrolase

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The enzyme appears in viruses and cellular organisms

EC NUMBER
COMMENTARY hide
3.5.4.42
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RECOMMENDED NAME
GeneOntology No.
N-isopropylammelide isopropylaminohydrolase
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REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
N-isopropylammelide + H2O = cyanuric acid + isopropylamine
show the reaction diagram
Involved in a pathway by which the herbicide atrazine, 2-chloro-4-(ethylamino)-6-(isopopylamino)-1,3,5-triazine, is degraded in bacteria via N-isopropylammelide, 2,4-dihydroxy-6-(isopropylamino)-1,3,5-triazine
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of C-N bond
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Atrazine degradation
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atrazine degradation I (aerobic)
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Metabolic pathways
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Microbial metabolism in diverse environments
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degradation of aromatic, nitrogen containing compounds
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SYSTEMATIC NAME
IUBMB Comments
N-isopropylammelide isopropylaminohydrolase
Requires Zn2+. This bacterial enzyme is involved in degradation of the herbicide atrazine. It can hydrolyse other N-substituted amino dihydroxy-s-triazine molecules, and prefers substrates with linear N-alkyl groups to those with branched alkyl groups.
CAS REGISTRY NUMBER
COMMENTARY hide
203810-02-0
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
synonym Pseudomonas sp. strain NRRLB-12227
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
isolated from a Thai soil, highly homologous to Arthrobacter histidinolovorans, gene atzC
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Manually annotated by BRENDA team
Flavobacterium oryzihabitans
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Manually annotated by BRENDA team
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Manually annotated by BRENDA team
strain SP12
SwissProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
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UniProt
Manually annotated by BRENDA team
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
physiological function
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the enzyme is involved in degradation of the herbicide atrazine
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
6-(propan-2-ylamino)-1,3,5-triazine-2,4-diol + H2O
1,3,5-triazine-2,4,6-triol + propan-2-amine
show the reaction diagram
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ir
ammelide + H2O
cyanuric acid + ammonia
show the reaction diagram
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?
ammelide + H2O
cyanuric acid + NH3
show the reaction diagram
N-cyclopropylammelide + H2O
cyanuric acid + cyclopropylamine
show the reaction diagram
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?
N-dimethylammelide + H2O
cyanuric acid + dimethylamine
show the reaction diagram
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?
N-ethylammelide + H2O
cyanuric acid + ethylamine
show the reaction diagram
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?
N-hydroxyethylammelide + H2O
cyanuric acid + 2-aminoethanol
show the reaction diagram
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?
N-isopropyl ammelide + H2O
cyanuric acid + propan-2-amine
show the reaction diagram
N-isopropylammelide + H2O
?
show the reaction diagram
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involved in a pathway by which the herbicide atrazine, 2-chloro-4-(ethylamino)-6-(isopopylamino)-1,3,5-triazine, is degraded in bacteria via N-isopropylammelide, 2,4-dihydroxy-6-(isopropylamino)-1,3,5-triazine
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N-isopropylammelide + H2O
cyanuric acid
show the reaction diagram
N-isopropylammelide + H2O
cyanuric acid + isopropylamine
show the reaction diagram
N-methylammelide + H2O
cyanuric acid + methylamine
show the reaction diagram
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?
N-tert-butylammelide + H2O
cyanuric acid + tert-butylamine
show the reaction diagram
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?
additional information
?
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the isolated strain is capable of degrading atrazine. Displacement of the three substituents on the s-triazine ring is mediated by three enzymatic steps encoded by the genes atzA, atzB, and atzC. AtzC stoichiometrically metabolizes N-isopropylammelide to cyanuric acid and N-isopropylamine
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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
6-(propan-2-ylamino)-1,3,5-triazine-2,4-diol + H2O
1,3,5-triazine-2,4,6-triol + propan-2-amine
show the reaction diagram
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ir
ammelide + H2O
cyanuric acid + NH3
show the reaction diagram
N-isopropyl ammelide + H2O
cyanuric acid + propan-2-amine
show the reaction diagram
N-isopropylammelide + H2O
?
show the reaction diagram
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involved in a pathway by which the herbicide atrazine, 2-chloro-4-(ethylamino)-6-(isopopylamino)-1,3,5-triazine, is degraded in bacteria via N-isopropylammelide, 2,4-dihydroxy-6-(isopropylamino)-1,3,5-triazine
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additional information
?
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the isolated strain is capable of degrading atrazine. Displacement of the three substituents on the s-triazine ring is mediated by three enzymatic steps encoded by the genes atzA, atzB, and atzC. AtzC stoichiometrically metabolizes N-isopropylammelide to cyanuric acid and N-isopropylamine
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Co2+
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completely restores activity of metal-depleted enzyme
Mn2+
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completely restores activity of metal-depleted enzyme
Ni2+
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completely restores activity of metal-depleted enzyme
Zn2+
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0.5 mol per mol of subunit
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1,10-phenanthroline
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5 mM, 30% residual activity, Mn2+, Co2+ or Ni2+ restores
2-amino-4-hydroxy-1,3,5-s-triazine
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i.e, 5-azacytosine, competitive
8-Hydroxyquinoline-5-sulfonic acid
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5 mM, 30% residual activity, Mn2+, Ni2+ or Co2+ restores
ammeline
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competitive
EDTA
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5 mM, about 10% loss of activity
N-ethylammeline
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N-hydroxyethylammeline
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competitve
N-isopropylammeline
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nitrate
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nitrate completely inhibits atrazine degradation activity in strain KU001
additional information
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ammonium and urea have no effect on atrazine degradation activity in strain KU001
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
citrate
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activates atrazine degradation activity in vivo
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.3 - 25
ammelide
0.58
N-cyclopropylammelide
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pH 7.6, 25C
0.155
N-dimethylammelide
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pH 7.6, 25C
0.308
N-ethylammelide
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pH 7.6, 25C
3.86
N-hydroxyethylammelide
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pH 7.6, 25C
0.406
N-isopropylammelide
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pH 7.6, 25C
0.817
N-methylammelide
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pH 7.6, 25C
0.299
N-tert-butylammelide
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pH 7.6, 25C
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
10.4 - 778.5
ammelide
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
7 - 140
ammelide
33646
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.015
2-amino-4-hydroxy-1,3,5-s-triazine
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pH 7.6, 25C
0.687
ammeline
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pH 7.6, 25C
0.129
N-hydroxyethylammeline
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pH 7.6, 25C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
16.2
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25C, pH 7.6
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
44000
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4 * 44000, SDS-PAGE, 4 * 44900, deduced from gene sequence
44900
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4 * 44000, SDS-PAGE, 4 * 44900, deduced from gene sequence
44940
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calculated from the sequence of the AtzC gene
174000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
structure of AtzC at 1.84 A with weak inhibitor malonate bound in the active site. Malonate adopts a planar conformation in the structure and packs against residue as well as interacting with Lys65, Gln160, His219 and Asn304. Enzyme is a dimer of dimers
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
57
melting temperature, mutant H249A
60
melting temperature, wild-type
63
melting temperature, mutant H219A
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
recombinant enzyme
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
cloned into pGem-T-EasyII plasmid
expression in Escherichia coli
gene atzB, cloned from soil isolated strain, DNA and amino acid sequence determination and analysis, sequence comparison
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gene atzB, DNA and amino acid sequence determination and analysis
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EXPRESSION
ORGANISM
UNIPROT
LITERATURE
atrazine degradation is not inhibited in cells grown on ammonium, nitrate, or urea, as compared with cells cultivated on growth-limiting nitrogen sources
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation
D188A
about 6fold increase in kcat/Km value
D188N
activity similar to wild-type
D303A
activity similar to wild-type
D303N
activity similar to wild-type
H219A
4fold increase in kcat value
H249A
substitution of an active site histidine residue, complete loss of activity
K65A
more than 15fold increase in kcat/Km value, 30fold increase in kcat for ammelide
K65R
more than 15fold increase in kcat/Km value, 12fold increase in kcat for ammelide
N304A
slight increase in kcat for ammelide
N304D
7fold increase in kcat for ammelide
Q160A
activity similar to wild-type
Q160E
60% reduction in kcat value
W309A
mutation opens up the area above the ring and increases the catalytic rate by almost a factor of two
W309F
slight reduction in catalytic rate
Renatured/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
activity of metal-depleted enzyme may be restored by Zn2+, Fe2+, Mn2+, Co2+, Ni2+
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation
molecular biology
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