Information on EC 3.5.1.84 - biuret amidohydrolase

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

EC NUMBER
COMMENTARY hide
3.5.1.84
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RECOMMENDED NAME
GeneOntology No.
biuret amidohydrolase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
biuret + H2O = urea-1-carboxylate + NH3
show the reaction diagram
involved in a pathway by which the herbicide atrazine, 2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine, is degraded in bacteria
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hydrolysis of linear amides
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PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
cyanurate degradation
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Atrazine degradation
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Metabolic pathways
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Microbial metabolism in diverse environments
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SYSTEMATIC NAME
IUBMB Comments
biuret amidohydrolase
Along with EC 3.5.2.15 (cyanuric acid amidohydrolase) and EC 3.5.1.54 (allophanate hydrolase), 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. Urea-1-carboxylate rather than urea (as was thought previously) is the 2-nitrogen intermediate in cyanuric-acid metabolism in bacteria [2]. The product, urea-1-carboxylate, can spontaneously decarboxylate under acidic conditions to form urea but, under physiological conditions, it can be converted into CO2 and ammonia by the action of EC 3.5.1.54 [2].
CAS REGISTRY NUMBER
COMMENTARY hide
95567-88-7
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Gordonia rubripertincta DSM 10347 / NRRLB-15444R
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Manually annotated by BRENDA team
Hormodendrum sp.
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Manually annotated by BRENDA team
Penicillium spp.
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
(E)-diazene-1,2-dicarboxamide + H2O
?
show the reaction diagram
1,1-dimethylurea + H2O
?
show the reaction diagram
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low specific activity
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?
1-carbamimidoylthiourea + H2O
?
show the reaction diagram
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low specific activity
-
-
?
1-ethylurea + H2O
?
show the reaction diagram
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low specific activity
-
-
?
1-formylurea + H2O
?
show the reaction diagram
1-hydroxyurea + H2O
?
show the reaction diagram
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low specific activity
-
-
?
1-methylthiourea + H2O
?
show the reaction diagram
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low specific activity
-
-
?
1-methylurea + H2O
?
show the reaction diagram
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low specific activity
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-
?
1-prop-2-en-1-ylurea + H2O
?
show the reaction diagram
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low specific activity
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-
?
2-cyanoacetamide + H2O
?
show the reaction diagram
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low specific activity
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-
?
2-methylpropanamide + H2O
?
show the reaction diagram
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low specific activity
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?
3-oxobutanamide + H2O
?
show the reaction diagram
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low specific activity
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-
?
acetamide + H2O
?
show the reaction diagram
biuret + H2O
allophanic acid + NH3
show the reaction diagram
biuret + H2O
urea + CO2 + NH3
show the reaction diagram
biuret + H2O
urea-1-carboxylate + NH3
show the reaction diagram
butanediamide + H2O
?
show the reaction diagram
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low specific activity
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?
carbamoylcarbamate + H2O
?
show the reaction diagram
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low specific activity
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?
dicarbonimidic diamide + H2O
?
show the reaction diagram
dicarbonodithioimidic diamide + H2O
?
show the reaction diagram
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low specific activity
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?
ethanediamide + H2O
?
show the reaction diagram
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low specific activity
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-
?
ethanethioamide + H2O
?
show the reaction diagram
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low specific activity
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?
ethyl acetate + H2O
?
show the reaction diagram
ethyl carbamoylcarbamate + H2O
?
show the reaction diagram
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low specific activity
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?
formamide + H2O
?
show the reaction diagram
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low specific activity
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?
hexanediamide + H2O
?
show the reaction diagram
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low specific activity
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?
hydrazinecarbothioamide + H2O
?
show the reaction diagram
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low specific activity
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?
hydrazinecarboxamide + H2O
?
show the reaction diagram
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low specific activity
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?
N-carbamothioylacetamide + H2O
?
show the reaction diagram
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low specific activity
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?
N-carbamoylacetamide + H2O
?
show the reaction diagram
N-hydroxydicarbonimidic diamide + H2O
?
show the reaction diagram
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low specific activity
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?
N-nitrodicarbonimidic diamide + H2O
?
show the reaction diagram
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low specific activity
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?
prop-2-enamide + H2O
?
show the reaction diagram
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low specific activity
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?
propanediamide + H2O
?
show the reaction diagram
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low specific activity
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?
sulfuric diamide + H2O
?
show the reaction diagram
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low specific activity
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?
thiourea + H2O
?
show the reaction diagram
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low specific activity
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?
urea + H2O
?
show the reaction diagram
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
biuret + H2O
allophanic acid + NH3
show the reaction diagram
biuret + H2O
urea + CO2 + NH3
show the reaction diagram
biuret + H2O
urea-1-carboxylate + NH3
show the reaction diagram
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
additional information
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the addition of ZnSO4 or CoSO4 (0.05 mM and 1 mM) to enzyme assays gives only nominal increases in activity. No metal cofactors are detected
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
DEAE-cellulose
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strain D, slow inactivation, inactivation accelerated markedly on addition of dithiothreitol
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dithiothreitol
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strain D, accelerates inactivation by DEAE-cellulose markedly
N-ethylmaleimide
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more than 90% loss of activity at 0.05 mM; more than 90% loss of activity at 0.5 mM
Tris
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strain D, inactivation
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.023 - 2.528
Biuret
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.07 - 11.9
Biuret
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.115 - 170
Biuret
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.02
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below 0.02 micromol/min/mg, using 1,1-dimethylurea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 1-carbamimidoylthiourea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 1-ethylurea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 1-hydroxyurea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 1-methylthiourea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 1-methylurea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 1-prop-2-en-1-ylurea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 2-cyanoacetamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 2-methylpropanamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using 3-oxobutanamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using acetamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using butanediamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using carbamoylcarbamate as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using dicarbonodithioimidic diamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using (E)-diazene-1,2-dicarboxamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using ethanediamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using ethanethioamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using ethyl acetate as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using ethyl carbamoylcarbamate as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using formamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using hexanediamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using hydrazinecarbothioamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using hydrazinecarboxamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using N-carbamothioylacetamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using N-hydroxydicarbonimidic diamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using N-nitrodicarbonimidic diamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using prop-2-enamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using propanediamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using sulfuric diamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using thiourea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C; below 0.02 micromol/min/mg, using urea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C
0.04
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using N-carbamoylacetamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C
0.17
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using 1-formylurea as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C
6.08
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using dicarbonimidic diamide as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C
8.9
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using biuret as substrate, in 0.1 M potassium phosphate buffer, pH 8.5, at 22°C
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 11
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about 50% activity at pH 4.5 and pH 11.0
additional information
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strain D, essentially inactive at pH 9.5
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
5.7
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calculated from amino acid sequence
PDB
SCOP
CATH
UNIPROT
ORGANISM
Pseudomonas sp. (strain ADP);
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
27121
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x * 27121, calculated from amino acid sequence
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
crystals suitable for X-ray analysis are grown using a combination of seeding, in situ proteolysis, formulation variation and additive screening. Structures of the enzyme, the enzyme with bound inhibitor and variants of the enzyme
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
potassium phosphate buffer yields optimal catalytic rates
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strain D, bovine serum albumin, large amounts, stabilizes, rapid loss of activity can be decreased to 25% with large amounts of bovine serum albumin
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strain D, dithiothreitol, 10 mM, stabilizes, rapid loss of activity can be decreased to 25% with 10 mM dithiothreitol
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strain D, enzyme very labile during purification
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strain D, enzyme very labile, complete loss of activity in a few hours
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strain D, glycerol, 1.5 M, stabilizes, rapid loss of activity can be decreased to 25% with 1.5 M glycerol
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
strain D, rapid complete loss of activity in a few hours
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
nickel affinity column chromatography
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partial, strain D, NRRLB-12228, enzyme very labile during purification
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expressed in Escherichia coli DH5alpha cells
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expression in Escherichia coli
strain 99, cloning large conjugal plasmid pPDL12, carrying the genes encoding the s-triazine degradation pathway, including the trzE gene encoding biuret amidohydrolase in Klebsiella planticola ATCC 33531, expression in Klebsiella planticola ATCC 33531
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C175S
the mutant enzyme binds biuret but is non-catalytic. The structure of the inactive C175S mutant enzyme with substrate bound in the active site reveals that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of enzyme variants
F41A
kcat/Km for buiret is 540fold lower than the wild-type value
F41L
kcat/Km for buiret is 713fold lower than the wild-type value
F41W
kcat/Km for buiret is 73fold lower than the wild-type value
F41Y
kcat/Km for buiret is 139fold lower than the wild-type value
K142A
kcat/Km for buiret is 297fold lower than the wild-type value
Q215A
kcat/Km for buiret is 419fold lower than the wild-type value
Q215E
kcat/Km for buiret is 1302fold lower than the wild-type value
Q215N
kcat/Km for buiret is 38fold lower than the wild-type value
C175S
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the mutant enzyme binds biuret but is non-catalytic. The structure of the inactive C175S mutant enzyme with substrate bound in the active site reveals that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of enzyme variants
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F41A
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kcat/Km for buiret is 540fold lower than the wild-type value
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F41L
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kcat/Km for buiret is 713fold lower than the wild-type value
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K142A
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kcat/Km for buiret is 297fold lower than the wild-type value
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Q215A
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kcat/Km for buiret is 419fold lower than the wild-type value
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APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture