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Information on EC 3.5.1.54 - allophanate hydrolase and Organism(s) Pseudomonas sp. and UniProt Accession Q936X2
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3.5.1.54 allophanate hydrolaseIUBMB Comments
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 .
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Word Map
- 3.5.1.54
- carboxylase
- ammonia
-
amidolyase
- amidase
- s-triazine
- dioxide
- ah
-
cyanuric
- biuret
-
ribonucleic
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kluyveromyces
- chlamydomonas
- enterobacter
- atrazine
- lactis
- allantoin
The taxonomic range for the selected organisms is: Pseudomonas sp.
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
The expected taxonomic range for this enzyme is: Bacteria, Eukaryota, Archaea
Synonyms
allophanate hydrolase, allophanate lyase, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
allophanate lyase
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
hydrolysis of linear amides
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-
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PATHWAY SOURCE
PATHWAYS
BRENDA
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
9076-72-6
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
malonamic acid + hydroxylamine
malonohydroxamate + ?
hydroxylamine trapping activity
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-
?
allophanate + H2O
2 CO2 + 2 NH3
the reverse reaction is catalyzed by urea carboxylase
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-
ir
allophanate + H2O
2 CO2 + 2 NH3
the N-terminal domain of allophanate hydrolase deaminates allophanate to produce ammonia and N-carboxycarbamate, while the smaller C-terminal domain seems not to be required for cataytic activity, overview
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-
?
allophanate + H2O
NH3 + CO2
the enzyme is involved in bacterial cyanuric acid metabolism
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-
ir
additional information
?
-
allophanate also shows nonenzymatic decomposition, half-life at pH 8.0 is 50 h
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-
?
additional information
?
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substrate specificity, no activity with methyl allophanate, hydantoic acid, oxamic acid, hydroxyurea, methyl carbamate, N-methylurea, acetylurea, 1-acetyl-2-thiourea, and semicarbazide, no activity with rhodanine, rhodanine-3-acetic acid, 3-aminorhodanine, (4R)-(-)-2-thioxo-4-thiazolidinecarboxylic acid, (-)-2-oxo-4-thiazolinecarboxylic acid, and 2-amino-5-bromothiazol
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-
?
additional information
?
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-
substrate specificity, no activity with methyl allophanate, hydantoic acid, oxamic acid, hydroxyurea, methyl carbamate, N-methylurea, acetylurea, 1-acetyl-2-thiourea, and semicarbazide, no activity with rhodanine, rhodanine-3-acetic acid, 3-aminorhodanine, (4R)-(-)-2-thioxo-4-thiazolidinecarboxylic acid, (-)-2-oxo-4-thiazolinecarboxylic acid, and 2-amino-5-bromothiazol
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?
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
allophanate + H2O
2 CO2 + 2 NH3
the reverse reaction is catalyzed by urea carboxylase
-
-
ir
allophanate + H2O
NH3 + CO2
the enzyme is involved in bacterial cyanuric acid metabolism
-
-
ir
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
Michaelis-Menten kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
metabolism
allophanate hydrolase also participates in the cyanuric acid mineralization pathway, in which the cyanuric acid ring is hydrolytically opened by cyanuric acid hydrolase (AtzD or TrzD, EC 3.5.2.15) forming the unstable metabolite carboxybiuret, which spontaneously decarboxylates to form biuret. Allophanate is produced from biuret by AtzE (biuret hydrolase; EC 3.5.1.84) via a single deamination. Hydrolysis of allophanate is then carried out by allophanate hydrolase. Both pathways, cyanuric acid mineralization pathway and urea carboxylase pathway, depend upon allophanate deamination by allophanate hydrolase to avoid spontaneous decarboxylation (and urea formation)
additional information
the N-terminal amidase domain of the enzyme reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea), structure analysis, overview. The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function. AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product. The positions of the amino acids essential for catalysis (Ser165, Ser189, and Lys91) and substrate binding (Tyr320 and Arg328), are highly conserved
evolution
allophanate hydrolase is a member of the amidase family of enzymes that possess a conserved serine- and glycine-rich motif, the so-called amidase signature sequence
evolution
the positions of the amino acids essential for catalysis (Ser165, Ser189, and Lys91) and substrate binding (Tyr320 and Arg328), are highly conserved
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
C-terminally truncated enzyme mutant, AtzF467, small-angle X-ray scattering
homotetramer
the full-length wild-type enzyme is a homotetramer in solution, small-angle X-ray scattering
additional information
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 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
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 20°C, X-ray diffraction structure determination and analysis at 2.5 A resolution, molecular replacement
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
H488A
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
additional information
additional information
construction of the gene encoding C-terminally truncated AtzF mutant, AtzF467
additional information
-
construction of the gene encoding C-terminally truncated AtzF mutant, AtzF467
additional information
construction of the gene encoding C-terminally truncated AtzF mutant, AtzF467, the mutant shows similar activity as the wild-type enzyme below pH 8.0, but slightly reduced activity above pH 8.0
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
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
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21(lambdaDE3)
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)
gene atzF, expression of the His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)
gene atzF, sequence comparisons, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(lambdaDE3)
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Cheng, G.; Shapir, N.; Sadowsky, M.J.; Wackett, L.P.
Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism
Appl. Environ. Microbiol.
71
4437-4445
2005
Agrobacterium tumefaciens, Agrobacterium tumefaciens J14a, Enterobacter cloacae (Q9ALV2), Enterobacter cloacae, Enterobacter cloacae 99 (Q9ALV2), Herbaspirillum huttiense, Herbaspirillum huttiense NRRL B-12228, Pseudomonas sp. (Q936X2), Ralstonia pickettii, Ralstonia pickettii D
Shapir, N.; Sadowsky, M.J.; Wackett, L.P.
Purification and characterization of allophanate hydrolase (AtzF) from Pseudomonas sp. strain ADP
J. Bacteriol.
187
3731-3738
2005
Pseudomonas sp. (Q936X2), Pseudomonas sp.
Balotra, S.; Newman, J.; French, N.G.; Briggs, L.J.; Peat, T.S.; Scott, C.
Crystallization and preliminary X-ray diffraction analysis of the amidase domain of allophanate hydrolase from Pseudomonas sp. strain ADP
Acta crystallogr. Sect. F
70
310-315
2014
Pseudomonas sp. (Q936X2), Pseudomonas sp.
Balotra, S.; Newman, J.; Cowieson, N.P.; French, N.G.; Campbell, P.M.; Briggs, L.J.; Warden, A.C.; Easton, C.J.; Peat, T.S.; Scott, C.
X-ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex
Appl. Environ. Microbiol.
81
470-480
2015
Pseudomonas sp. (Q936X2)
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