BRENDA - Enzyme Database
show all sequences of 3.5.1.54

The structure of allophanate hydrolase from Granulibacter bethesdensis provides insights into substrate specificity in the amidase signature family

Lin, Y.; St Maurice, M.; Biochemistry 52, 690-700 (2013)

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
gene CGDNIH1, expression of N-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain HMS174 (DE3)
Granulibacter bethesdensis
Crystallization (Commentary)
Crystallization
Organism
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
Granulibacter bethesdensis
Engineering
Amino acid exchange
Commentary
Organism
R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299A/R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299A/R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299F/R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299F/R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetics of wild-type and mutant enzymes, overview
Granulibacter bethesdensis
0.1
-
allophanate
pH 7.4, 25°C, recombinant wild-type enzyme
Granulibacter bethesdensis
Molecular Weight [Da]
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
65000
-
x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE
Granulibacter bethesdensis
Natural Substrates/ Products (Substrates)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
allophanate + H2O
Granulibacter bethesdensis
-
2 CO2 + 2 NH3
-
-
?
allophanate + H2O
Granulibacter bethesdensis ATCC BAA-1260
-
2 CO2 + 2 NH3
-
-
?
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Granulibacter bethesdensis
Q0BRB0
gene CGDNIH1
-
Granulibacter bethesdensis ATCC BAA-1260
Q0BRB0
gene CGDNIH1
-
Purification (Commentary)
Commentary
Organism
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
Granulibacter bethesdensis
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
allophanate + H2O
-
733342
Granulibacter bethesdensis
2 CO2 + 2 NH3
-
-
-
?
allophanate + H2O
the enzyme exhibits high specificity for allophanate
733342
Granulibacter bethesdensis
2 CO2 + 2 NH3
-
-
-
?
allophanate + H2O
-
733342
Granulibacter bethesdensis ATCC BAA-1260
2 CO2 + 2 NH3
-
-
-
?
allophanate + H2O
the enzyme exhibits high specificity for allophanate
733342
Granulibacter bethesdensis ATCC BAA-1260
2 CO2 + 2 NH3
-
-
-
?
Subunits
Subunits
Commentary
Organism
?
x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE
Granulibacter bethesdensis
Temperature Optimum [°C]
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Granulibacter bethesdensis
Turnover Number [1/s]
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
18.2
-
allophanate
pH 7.4, 25°C, recombinant wild-type enzyme
Granulibacter bethesdensis
pH Optimum
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.4
-
assay at
Granulibacter bethesdensis
Cloned(Commentary) (protein specific)
Commentary
Organism
gene CGDNIH1, expression of N-terminally His8-tagged wild-type and mutant enzymes in Escherichia coli strain HMS174 (DE3)
Granulibacter bethesdensis
Crystallization (Commentary) (protein specific)
Crystallization
Organism
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
Granulibacter bethesdensis
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299A/R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299A/R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299F/R307A
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
Y299F/R307M
site-directed mutagenesis, the mutant shows highly reduced activity compared to the wild-type enzyme
Granulibacter bethesdensis
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
additional information
-
additional information
kinetics of wild-type and mutant enzymes, overview
Granulibacter bethesdensis
0.1
-
allophanate
pH 7.4, 25°C, recombinant wild-type enzyme
Granulibacter bethesdensis
Molecular Weight [Da] (protein specific)
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
65000
-
x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE
Granulibacter bethesdensis
Natural Substrates/ Products (Substrates) (protein specific)
Natural Substrates
Organism
Commentary (Nat. Sub.)
Natural Products
Commentary (Nat. Pro.)
Organism (Nat. Pro.)
Reversibility
allophanate + H2O
Granulibacter bethesdensis
-
2 CO2 + 2 NH3
-
-
?
allophanate + H2O
Granulibacter bethesdensis ATCC BAA-1260
-
2 CO2 + 2 NH3
-
-
?
Purification (Commentary) (protein specific)
Commentary
Organism
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
Granulibacter bethesdensis
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
allophanate + H2O
-
733342
Granulibacter bethesdensis
2 CO2 + 2 NH3
-
-
-
?
allophanate + H2O
the enzyme exhibits high specificity for allophanate
733342
Granulibacter bethesdensis
2 CO2 + 2 NH3
-
-
-
?
allophanate + H2O
-
733342
Granulibacter bethesdensis ATCC BAA-1260
2 CO2 + 2 NH3
-
-
-
?
allophanate + H2O
the enzyme exhibits high specificity for allophanate
733342
Granulibacter bethesdensis ATCC BAA-1260
2 CO2 + 2 NH3
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
?
x * 65000, about, recombinant His8-tagged enzyme, SDS-PAGE
Granulibacter bethesdensis
Temperature Optimum [°C] (protein specific)
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
25
-
assay at
Granulibacter bethesdensis
Turnover Number [1/s] (protein specific)
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
18.2
-
allophanate
pH 7.4, 25°C, recombinant wild-type enzyme
Granulibacter bethesdensis
pH Optimum (protein specific)
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
7.4
-
assay at
Granulibacter bethesdensis
General Information
General Information
Commentary
Organism
evolution
the enzyme belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cisSer-Lys catalytic triad
Granulibacter bethesdensis
metabolism
allophanate hydrolase catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2
Granulibacter bethesdensis
additional information
Tyr299 and Arg307 seem to serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172, nucleophilic attack by serine results in a covalent tetrahedral intermediate that is stabilized by an oxyanion hole. After displacement of ammonia, the covalent intermediate is hydrolyzed to release the product. The unique C-terminal domain is conserved, but it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Active site architecture and structure-function analysis, overview
Granulibacter bethesdensis
General Information (protein specific)
General Information
Commentary
Organism
evolution
the enzyme belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cisSer-Lys catalytic triad
Granulibacter bethesdensis
metabolism
allophanate hydrolase catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2
Granulibacter bethesdensis
additional information
Tyr299 and Arg307 seem to serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172, nucleophilic attack by serine results in a covalent tetrahedral intermediate that is stabilized by an oxyanion hole. After displacement of ammonia, the covalent intermediate is hydrolyzed to release the product. The unique C-terminal domain is conserved, but it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Active site architecture and structure-function analysis, overview
Granulibacter bethesdensis
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.0000017
-
allophanate
pH 7.4, 25°C, mutant R307A
Granulibacter bethesdensis
0.0000036
-
allophanate
pH 7.4, 25°C, mutant R307M
Granulibacter bethesdensis
0.0000071
-
allophanate
pH 7.4, 25°C, mutant Y299A/R307M
Granulibacter bethesdensis
0.000019
-
allophanate
pH 7.4, 25°C, mutant Y299F/R307M
Granulibacter bethesdensis
0.000027
-
allophanate
pH 7.4, 25°C, mutant Y299A/R307A
Granulibacter bethesdensis
0.0024
-
allophanate
pH 7.4, 25°C, mutant Y299A
Granulibacter bethesdensis
0.0035
-
allophanate
pH 7.4, 25°C, mutant Y299F
Granulibacter bethesdensis
182
-
allophanate
pH 7.4, 25°C, recombinant wild-type enzyme
Granulibacter bethesdensis
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.0000017
-
allophanate
pH 7.4, 25°C, mutant R307A
Granulibacter bethesdensis
0.0000036
-
allophanate
pH 7.4, 25°C, mutant R307M
Granulibacter bethesdensis
0.0000071
-
allophanate
pH 7.4, 25°C, mutant Y299A/R307M
Granulibacter bethesdensis
0.000019
-
allophanate
pH 7.4, 25°C, mutant Y299F/R307M
Granulibacter bethesdensis
0.000027
-
allophanate
pH 7.4, 25°C, mutant Y299A/R307A
Granulibacter bethesdensis
0.0024
-
allophanate
pH 7.4, 25°C, mutant Y299A
Granulibacter bethesdensis
0.0035
-
allophanate
pH 7.4, 25°C, mutant Y299F
Granulibacter bethesdensis
182
-
allophanate
pH 7.4, 25°C, recombinant wild-type enzyme
Granulibacter bethesdensis
Other publictions for EC 3.5.1.54
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
733179
Balotra
X-ray structure of the amidase ...
Pseudomonas sp.
Appl. Environ. Microbiol.
81
470-480
2015
-
-
1
1
2
-
-
1
-
-
-
1
-
3
-
-
1
-
-
-
-
-
2
3
1
1
-
-
1
-
-
-
-
-
-
-
-
1
-
1
2
-
-
-
-
1
-
-
-
1
-
-
-
1
-
-
-
-
2
3
1
1
-
-
1
-
-
-
-
2
2
-
-
-
733034
Balotra
Crystallization and preliminar ...
Pseudomonas sp.
Acta crystallogr. Sect. F
70
310-315
2014
-
-
1
1
1
-
-
-
-
-
2
1
-
3
-
-
1
-
-
-
-
-
2
1
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1
-
1
1
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2
1
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1
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
2
2
-
-
-
733342
Lin
The structure of allophanate h ...
Granulibacter bethesdensis, Granulibacter bethesdensis ATCC BAA-1260
Biochemistry
52
690-700
2013
-
-
1
1
8
-
-
2
-
-
1
2
-
3
-
-
1
-
-
-
-
-
4
1
1
-
-
1
1
-
-
-
-
-
-
-
-
1
-
1
8
-
-
-
-
2
-
-
1
2
-
-
-
1
-
-
-
-
4
1
1
-
-
1
1
-
-
-
-
3
3
-
8
8
734220
Fan
Structure and function of allo ...
Kluyveromyces lactis
J. Biol. Chem.
288
21422-21432
2013
-
-
1
1
3
-
-
-
-
-
-
-
-
3
-
-
1
1
-
-
-
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-
1
-
-
-
-
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1
-
1
3
-
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-
1
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
2
2
-
-
-
718475
Jacques
The structure of TTHA0988 from ...
no activity in Thermus thermophilus
Acta Crystallogr. Sect. D
67
105-111
2011
-
-
-
-
-
-
-
-
-
-
-
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-
3
-
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667267
Shapir
Purification and characterizat ...
Enterobacter cloacae, Enterobacter cloacae 99
Appl. Environ. Microbiol.
72
2491-2495
2006
-
-
1
-
-
-
1
1
-
1
3
2
-
6
-
-
-
-
-
-
1
-
10
1
1
-
-
1
1
-
-
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1
-
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1
-
1
-
1
3
2
-
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-
-
-
1
-
10
1
1
-
-
1
1
-
-
-
-
-
-
-
-
-
667259
Cheng
Allophanate hydrolase, not ure ...
Agrobacterium tumefaciens, Agrobacterium tumefaciens J14a, Enterobacter cloacae, Enterobacter cloacae 99, Herbaspirillum huttiense, Herbaspirillum huttiense NRRL B-12228, Pseudomonas sp., Ralstonia pickettii, Ralstonia pickettii D
Appl. Environ. Microbiol.
71
4437-4445
2005
-
-
4
-
-
-
-
-
-
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-
9
-
18
-
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19
-
1
-
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1
-
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4
-
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9
-
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-
-
-
-
19
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
668661
Kanamori
Allophanate hydrolase of Oleom ...
Oleomonas sagaranensis, Oleomonas sagaranensis HD-1
FEMS Microbiol. Lett.
245
61-65
2005
-
-
1
-
-
-
-
1
-
-
2
2
-
4
-
-
1
1
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-
1
-
4
1
1
-
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1
-
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1
-
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-
-
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-
1
-
-
2
2
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-
-
1
-
-
1
-
4
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
669098
Shapir
Purification and characterizat ...
Pseudomonas sp.
J. Bacteriol.
187
3731-3738
2005
-
-
1
-
3
-
1
2
-
-
2
1
-
4
-
-
1
-
-
-
1
-
6
1
-
-
-
2
1
1
-
-
1
-
-
-
-
1
-
-
3
-
-
1
1
2
-
-
2
1
-
-
-
1
-
-
1
-
6
1
-
-
-
2
1
1
-
-
-
-
-
-
-
-
209193
Nishiya
-
Production of urea amidolase b ...
Cyberlindnera jadinii, Cyberlindnera jadinii CA (u)-37
Seibutsu Shiro Bunseki
18
288-293
1995
-
-
-
-
-
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2
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1
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1
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-
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-
-
-
-
-
-
-
1609
Sumrada
Urea carboxylase and allophana ...
Saccharomyces cerevisiae
J. Biol. Chem.
257
9119-9127
1982
-
-
-
-
-
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-
1
1
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3
-
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1
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1
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1
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2
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209196
Maitz
-
Purification and properties of ...
Chlamydomonas reinhardtii
Biochim. Biophys. Acta
714
486-491
1982
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5
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2
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5
1
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1
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1
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1
1
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1602
Whitney
Urea carboxylase and allophana ...
Saccharomyces cerevisiae
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49
45-51
1972
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