Information on EC 2.3.1.183 - phosphinothricin acetyltransferase

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

EC NUMBER
COMMENTARY hide
2.3.1.183
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RECOMMENDED NAME
GeneOntology No.
phosphinothricin acetyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
acetyl-CoA + phosphinothricin = CoA + N-acetylphosphinothricin
show the reaction diagram
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-
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REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Acyl group transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
Biosynthesis of antibiotics
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phosalacine biosynthesis
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phosphinothricin tripeptide biosynthesis
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Phosphonate and phosphinate metabolism
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SYSTEMATIC NAME
IUBMB Comments
acetyl-CoA:phosphinothricin N-acetyltransferase
The substrate phosphinothricin is used as a nonselective herbicide and is a potent inhibitor of EC 6.3.1.2, glutamate---ammonia ligase, a key enzyme of nitrogen metabolism in plants [2].
CAS REGISTRY NUMBER
COMMENTARY hide
111069-93-3
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
an improved transformation method for Beauveria bassiana is developed, therefore a new expression vector carrying the bar gene as a marker is constructed, the bar gene is originally isolated from Streptomyces hygroscopicus and Streptomyces viridochromogenes
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Manually annotated by BRENDA team
the bar gene is originally isolated from Streptomyces hygroscopicus and Streptomyces viridochromogenes
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-
Manually annotated by BRENDA team
transgenic plants
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Manually annotated by BRENDA team
strain AB2253, soil isolate
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Manually annotated by BRENDA team
soil isolate
TREMBL
Manually annotated by BRENDA team
putative uncharacterized protein
UniProt
Manually annotated by BRENDA team
strain DX-35
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-
Manually annotated by BRENDA team
strain FX-90
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Manually annotated by BRENDA team
strain FX-90
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Manually annotated by BRENDA team
strain AB3534, soil isolate, DDBJ no. DD355820
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-
Manually annotated by BRENDA team
strain Tü494
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Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
metabolism
detoxification of the nonselective herbicide phosphinothricin, commercial named Roundup Ready, Finale or Basta
physiological function
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
acetyl-CoA + 5,5'-dithiobis-(2-nitrobenzoic acid)
CoA + 5-thio-2-nitrobenzoic acid
show the reaction diagram
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-
-
?
acetyl-CoA + bialaphos
CoA + L-N-acetylphosphinothricin bialophos
show the reaction diagram
acetyl-CoA + L-methionine sulfone
?
show the reaction diagram
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-
-
?
acetyl-CoA + L-methionine sulfoximine
?
show the reaction diagram
acetyl-CoA + L-phosphinothricin
CoA + L-N-acetylphosphinothricin
show the reaction diagram
acetyl-CoA + methionine sulfone
CoA + ?
show the reaction diagram
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-
-
?
acetyl-CoA + phosphinothricin
CoA + N-acetylphosphinothricin
show the reaction diagram
phosphinothricin + acetyl-CoA
L-N-acetylphosphinothricin + CoA
show the reaction diagram
additional information
?
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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
acetyl-CoA + bialaphos
CoA + L-N-acetylphosphinothricin bialophos
show the reaction diagram
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-
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?
acetyl-CoA + L-phosphinothricin
CoA + L-N-acetylphosphinothricin
show the reaction diagram
acetyl-CoA + phosphinothricin
CoA + N-acetylphosphinothricin
show the reaction diagram
additional information
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
acetyl-CoA
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
aminooxyacetic acid
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ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
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the enzyme activity is inducible 10fold by L-phosphinothricin
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.03
acetyl-CoA
apparent KM
1.3
L-Methionine sulfone
apparent KM
0.156 - 1.3
L-methionine sulfoximine
0.076 - 2.2
L-phosphinothricin
14.31
methionine sulfone
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additional information
additional information
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
610
L-Methionine sulfone
Pseudomonas aeruginosa
Q9HUU7
apparent Kcat
0.5 - 505
L-methionine sulfoximine
2.18
L-phosphinothricin
Rhodococcus sp.
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pH 8.0, 35°C
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.0177
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at 30°C, enzyme activity in crude extract of the transgenic soybean line h-7, in wild-type plants no activity is detectable
0.02
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induced activity, cell extract
0.0212
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strain DX-35, cell extract
0.0221
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at 30°C, enzyme activity in crude extract of the transgenic soybean line m-12, in wild-type plants no activity is detectable
0.0357
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at 30°C, enzyme activity in crude extract of the transgenic soybean line h-9, in wild-type plants no activity is detectable
additional information
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.2
activity assay
8.5
in Tris buffer
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
6 - 9
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no activity at pH 5.0 and pH 10.0
7 - 9.5
Catalytic activity reaches a maximum at pH 8 to pH 8.5. In Tris buffer at pH 7 and pH 9.5 20-30% catalytic activity remains. In phosphate buffer, the activity at pH 7 and pH 6.5 is 15% and 10%, respectively. At pH 6 the enzyme shows no activity.
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0 - 10
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more than 50% of maximum activity
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
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anthesis
Manually annotated by BRENDA team
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low level expression
Manually annotated by BRENDA team
additional information
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20000
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2 * 20000, about, recombinant enzyme
20621
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x * 20621, nucleotide sequence calculation
28000
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determined by SDS-PAGE and Western Blot analysis
40000
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recombinant enzyme, gel filtration
44000
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x * 44000, recombinant GST-fusion protein
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a high-resolution, 1.55 A, crystal structure of pita in complex with L-methionine sulfoximine is solved
molecular modeling of structure. Enzyme is a dimer. Ring stacking and a salt bridge derived from residues His114-Trp152 and Arg72-Glu82, respectively, are important forces for the subunit interaction
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TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
30
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80 min, more than 80% residual activity
50
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100 min, 60% residual activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
The degradability of purificated PAT obtained from Escherichia coli in simulated gastric fluid is clearly apparent within at least 30 s. However, PAT degradation in leaf tissue powder from zoysia grass is significantly delayed, but complete within at least 5 min.
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
for the purification of PAT from Pseudomonas fluorescens ammonium sulfate precipitation, a Source 15 Phenyl XK hydrophobic interaction column, and a Superdex 75 XK gel filtration column are used; recombinant enzyme from Pseudomonas fluorescens to homogeneity by ammonium sulfate fractionation, hydrophobic interaction chromatography, and gel filtration
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recombinant enzyme from Escherichia coli by ammonium sulfate fractionation, anion exchange, hydrophobic interaction, and hydroxylapatite chromatography, followed by gel filtration, to about 90% purity
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recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, recombinant enzyme from transgenic maize and rape plants by immunoaffinity chromatography, method development, overview
simple, time-saving, inexpensive and reproducible purification method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the phosphinothricin acetyltransferase protein, which can then be specifically released by acetyl-CoA. Phosphinothricin acetyltransferase protein can be purified to homogeneity from cottonseed with high recovery efficiency and enzymatically active
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the recombinant protein is purified by Ni2+ affinity chromatography and used for the production of polyclonal antibodies, for Western blot analysis the protein is extracted from peppers
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
a linear template for in vitro transcription and translation is created, the PAT gene is cloned into the vector pCR2.1TOPO, verified and subcloned into pET280 and pDOW1169 for expression in Escherichia coli or Pseudomonas fluorescens, respectively; overexpression in Escherichia coli Bl21(DE3) and Pseudomonas fluorescens strain DC454 requiring modification of the 5' end of the pat ORF, in vitro transcription and translation, optimization of the expression method, Pseudomonas fluorescens is the better host, the secondary structures in the 5' coding region of pat gene influences the expression efficiency, overview
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expressed as chimeric protein in Escherichia coli and in pea chloroplasts after transformation with Agrobacterium tumefaciens
expressed in Escherichia coli and in genetically modified zoysia grass Zoysia matrella
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expressed in Festuca pratensis after transformation with Agrobacterium tumefaciens
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expressed in Quercus suber after transformation with Agrobacterium tumefaciens
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expressed in soybean after transformation with Agrobacterium tumefaciens
expression in Escherichia coli
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gene bar, DNA and amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain BL21(DE3), expression in transgenic maize and rape plant leaves
gene bar, expression in Escherichia coli with gene fragments fused to the 3' end, the fusion gene product shows unaltered kinetic properties compared to the unmodified gene product
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gene bar, subcloning in Escherichia coli, functional expression in Nicotiana tabacum protoplasts using the Agrobacterium tumefaciens transfection system and The S35 CMV vector
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gene bar, transformation of and expression in chloroplasts of Nicotiana tabacum cv. Havana, reciprocal crosses between wild type and transplastomic tobacco plants confirms maternal inheritance of the PPT resistance and high levels of PAT activity in the transplastomic plants, phenotype, overview
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gene pat, DNA and amino acid sequence determination and analysis, translation of pat is initiated by a GTG codon, functional expression in transgenic Nicotiana tabacum plants by Agrobacterium tumefaciens strain LBA4404-mediated leaf-disc transformation replacing the GTG start codon by ATG, expression of wild-type and mutant enzymes in Streptomyces lividans strain TK23 and in Escherichia coli strains JM83 and S17.1, the gene confers resistance to L-phosphinothricin
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gene pat, expression in Capsicum annuum var. Subicho through Agrobacterium-mediated transformation, expression is highest in leaves, followed by stems, pollen, fruits, and lowest in roots
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gene pat, expression in Nicotiana tabacum var. W38 plant leaves and roots using the root-specific pat promoter of the hemoglobin gene from Parasponia andersonii, the transgenic tobacco plants show herbicide tolerance, overview
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gene pat, expression in transgenic Nicotiana tabacum, Daucus carota, and Medicago sativa plants, the functional expression of gene pat confers resistance to the herbicide L-phosphinothricin
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gene pat, functional expression in transgenic Nicotiana tabacum and Daucus carota plant leaves using the Agrobacterium tumefaciens strain LBA4404 transfection of leaves and direct gene transfer to protoplasts, respectively, subcloning in Escherichia coli strain S 17. I, the transgenic plants are resistant to L-phosphinothricin
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gene PttR, cloning of the gene from an phosphinothricin-tripeptide-resistant mutant, DNA and amino acid sequence determination and analysis, promoter analysis, expression in Streptomyces lividans strain TK23 and Escherichia coli strain JM83 using different plasmids, overview, the gene confers resistance to L-phosphinothricin
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into the pQE31 vector for expression in Escherichia coli M15 cells
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into the vector pBFT, the new construct contains the marker genes for phosphinothricin acetyltransferase and green fluorescence protein as a fusion sequence bar::egfp
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into the vector pET24a for expression in Escherichia coli BL21DE3 cells
overexpression in Escherichia coli
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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Depp4
mutant, in which pita is inactivated through an in-frame deletion
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
agriculture
analysis
biotechnology
molecular biology
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the bar gene represents a selectable and assayable reporter gene especially suitable for 3’-terminal gene fusions
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