Information on EC 3.5.1.54 - allophanate hydrolase

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

EC NUMBER
COMMENTARY hide
3.5.1.54
-
RECOMMENDED NAME
GeneOntology No.
allophanate hydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
urea-1-carboxylate + H2O = 2 CO2 + 2 NH3
show the reaction diagram
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of linear amides
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-
-
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Arginine biosynthesis
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Atrazine degradation
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cyanurate degradation
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Metabolic pathways
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Microbial metabolism in diverse environments
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urea degradation I
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urea cycle
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SYSTEMATIC NAME
IUBMB Comments
urea-1-carboxylate amidohydrolase
Along with EC 3.5.2.15 (cyanuric acid amidohydrolase) and EC 3.5.1.84 (biuret amidohydrolase), this enzyme forms part of the cyanuric-acid metabolism pathway, which degrades s-triazide herbicides, such as atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine], in bacteria. The yeast enzyme (but not that from green algae) also catalyses the reaction of EC 6.3.4.6, urea carboxylase, thus bringing about the hydrolysis of urea to CO2 and NH3 in the presence of ATP and bicarbonate. The enzyme from Pseudomonas sp. strain ADP has a narrow substrate specificity, being unable to use the structurally analogous compounds urea, hydroxyurea or methylcarbamate as substrate [6].
CAS REGISTRY NUMBER
COMMENTARY hide
9076-72-6
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain J14a, gene atzF
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Manually annotated by BRENDA team
strain J14a, gene atzF
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Manually annotated by BRENDA team
strain CA (u)-37
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-
Manually annotated by BRENDA team
strain CA (u)-37
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-
Manually annotated by BRENDA team
gene CGDNIH1
UniProt
Manually annotated by BRENDA team
strain NRRLB-12228, gene atzF
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-
Manually annotated by BRENDA team
strain NRRLB-12228, gene atzF
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Manually annotated by BRENDA team
no activity in Thermus thermophilus
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-
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Manually annotated by BRENDA team
strain HD-1, isolated from an oil field in Sagara, Japan
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Manually annotated by BRENDA team
strain HD-1, isolated from an oil field in Sagara, Japan
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Manually annotated by BRENDA team
strain D, gene atzF
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Manually annotated by BRENDA team
strain D, gene atzF
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
evolution
metabolism
physiological function
allophanate hydrolase is essential for urea utilization. The enzyme also has important functions in the eukaryotic pyrimidine nucleic acid precursor degradation pathway, the yeast-hypha transition that several pathogens utilize to escape the host defense, and an s-triazine herbicide degradation pathway in soil bacteria
additional information
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
allophanate + H2O
2 CO2 + 2 NH3
show the reaction diagram
allophanate + H2O
CO2 + NH3
show the reaction diagram
allophanate + H2O
NH3 + CO2
show the reaction diagram
biuret + H2O
?
show the reaction diagram
malonamic acid + H2O
?
show the reaction diagram
malonamic acid + hydroxylamine
malonohydroxamate + ?
show the reaction diagram
-
hydroxylamine trapping activity
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-
?
malonamide + 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
allophanate + H2O
2 CO2 + 2 NH3
show the reaction diagram
allophanate + H2O
CO2 + NH3
show the reaction diagram
allophanate + H2O
NH3 + CO2
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
5,5'-dithiobis(2-nitrobenzoic acid)
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acetoacetic acid
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Ki 0.3 mM
NEM
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allophanate protects
oxaloacetic acid
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phenyl phosphorodiamidate
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.042 - 1.5
allophanate
5.3
malonamic acid
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pH 9.5, recombinant enzyme
additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
6 - 18.2
allophanate
0.1
malonamic acid
Pseudomonas sp.
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pH 9.5, recombinant enzyme
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000017 - 182
allophanate
8043
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.3
acetoacetic acid
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3.2
phenyl phosphorodiamidate
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pH 9.5, recombinant enzyme
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.3
purified recombinant enzyme
108
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purified recombinant enzyme, substrate allophanate
additional information
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 9.5
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assay at
9.5
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recombinant enzyme
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7 - 10
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pH 7.0: about 65% of maximal activity, pH 10.0: about 30% of maximal activity
8 - 11.3
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recombinant enzyme
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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assay at
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
28 - 45
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maximal activity at 28C, 50% of maximal activity at 45C
PDB
SCOP
CATH
ORGANISM
UNIPROT
Granulibacter bethesdensis (strain ATCC BAA-1260 / CGDNIH1)
Granulibacter bethesdensis (strain ATCC BAA-1260 / CGDNIH1)
Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37)
Kluyveromyces lactis (strain ATCC 8585 / CBS 2359 / DSM 70799 / NBRC 1267 / NRRL Y-1140 / WM37)
Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
50000
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6 * 50000, recombinant enzyme, SDS-PAGE
61999
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2 * 61999, sequence calculation
65000
x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE
65401
2 * 68000, His-tagged recombinant enzyme, SDS-PAGE, 2 * 65401, sequence calculation
66223
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4 * 66223, sequence calculation
68000
2 * 68000, His-tagged recombinant enzyme, SDS-PAGE, 2 * 65401, sequence calculation
100000
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gel filtration
138000
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gel filtration, recombinant enzyme
144000
His-tagged recombinant enzyme, gel filtration
204000
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sedimentation analysis
260000
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gel filtration, recombinant enzyme
360000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homohexamer
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6 * 50000, recombinant enzyme, SDS-PAGE
homotetramer
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the full-length wild-type enzyme is a homotetramer in solution, small-angle X-ray scattering
tetramer
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4 * 66223, sequence calculation
additional information
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AtzF has two main domains: the catalytic domain and a second all-alpha-helical domain that forms the dimer interface. The C-terminal domain has a function in coordinating the quaternary structure of the enzyme. AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
purified enzyme free or in complex with the substrate analog malonate, hanging-drop vapor diffusion method, mixing of 4 mg/ml protein in 10 mM HEPES, pH 8.0, 50 mM NaCl, and 1 mM DTT, with reservoir solution in a 1:1 ratio to a final volume of 0.005 ml, the latter containing 100 mM PIPES, pH 6.5, and 1.04 M sodium malonate, room temperature, 2 months, followed by microseeding, 5-15 days, X-ray diffraction structure determination and analysis at 2.2-2.8 A resolution, molecular replacement
purified recombinant wild-type and mutant His-tagged SeMet-substituted enzymes, sitting drop vapour diffusion method, mixing of 10 mg/ml protein in 20 mM Tris/HCl, pH 7.5, 200 mM NaCl, 2-10 mM DTT, with reservoir containing 16% PEG 8000, 20% glycerol, and 0.04 M potassium phosphate, and 100 mM sodium/potassium tartrate, 20C, X-ray diffraction structure determination and analysis at 2.5-2.6 A resolution, molecular replacement
purified full-length wild-type enzyme and truncated mutant enzyme, trypsin-treated AtzF (in situ proteolysis) from 1 M ammonium sulfate, 1 M lithium sulfate, 0.1 M Tris-HCl, pH 8.5, AtzF467 crystals grown from 20% w/v PEG 6000, 0.1 M Na MES pH 6.5, 0.2 M calcium chloride, are used in microseeding for truncated AtzF crystal growth from 11% w/v PEG 3350, 2% Tacsimate, pH 5.0, X-ray diffraction structure determination and analysis at 2.5 A resolution
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purified recombinant wild-type and mutant enzymes, from a reservoir containing 11 to 14% w/v PEG 3350 and 2% Tacsimate reagent, pH 5.0, at 20C, X-ray diffraction structure determination and analysis at 2.5 A resolution, molecular replacement
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
as a component of urea amidolase, allophanate hydrolase domain inactivated, urea carboxylase activity detected
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component of a multienzyme complex, identical, multifunctional subunits
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recombinant active His-tagged enzyme from Escherichia coli strain BL21 (lambdaDE3) by nickel affinity chromatography and gel filtration to about 98% purity and apparent homogeneity
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recombinant enzyme from Escherichia coli strain BL21(DE3) by ultracentrifucation, ion exchange chromatography, and gel filtration, 2.8fold to homogeneity
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recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
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recombinant N-terminally His8-tagged wild-type and mutant enzymes from Escherichia coli strain HMS174 (DE3) by nickel affinity chromatography, dialysis, and anion exchange chromatography
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(lambdaDE3)
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recombinant wild-type and mutant His-tagged SeMet-substituted enzymes from Escherichia coli strain BL21 Star(DE3) by nickel affinity chromatography and gel filtration
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
gene atzF, DNA and amino acid sequence determination and analysis, recombinant expression of functional His-tagged enzyme in Escherichia coli strain BL21 (lambdaDE3)
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gene atzF, expression of the His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
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gene atzF, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(lambdaDE3)
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gene CGDNIH1, expression of N-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain HMS174 (DE3)
gene trzF, overexpression of His-tagged enzyme in presence of a chaperone in Escherichia coli strain BL21(DE3)
overexpression in Escherichia coli strain BL21(DE3)
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recombinant expression of wild-type and mutant His-tagged SeMet-substituted enzymes in Escherichia coli strain BL21 Star(DE3)
sequence analysis and comparison, and phylogenetic analysis
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Y299A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y299A/R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Y299A/R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Y299F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Y299F/R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Y299F/R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
R307A
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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R307M
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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Y299A
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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Y299A/R307M
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site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
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Y299F
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site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
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G559E/G572E
site-directed mutagenesis, crystal structure analysis, the mutant shows 14fold increaed Km for allophanate, and reduced substrate binding at the N-domain active site, but is catalytically active
S177A
site-directed mutagenesis, crystal structure analysis
H488A
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site-directed mutagenesis, the mutant shows similar activity as the wild-type enzyme below pH 8.0, but slightly reduced activity above pH 8.0
K91A
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site-directed mutagenesis, inactive mutant
S165A
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site-directed mutagenesis, inactive mutant
S189A
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site-directed mutagenesis, inactive mutant
additional information
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