Information on EC 2.3.2.15 - glutathione gamma-glutamylcysteinyltransferase

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

EC NUMBER
COMMENTARY hide
2.3.2.15
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RECOMMENDED NAME
GeneOntology No.
glutathione gamma-glutamylcysteinyltransferase
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
glutathione + [Glu(-Cys)]n-Gly = Gly + [Glu(-Cys)]n+1-Gly
show the reaction diagram
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-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
gamma-glutamylcysteinyl transfer
PATHWAY
BRENDA Link
KEGG Link
MetaCyc Link
phytochelatins biosynthesis
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SYSTEMATIC NAME
IUBMB Comments
glutathione:poly(4-glutamyl-cysteinyl)glycine 4-glutamylcysteinyltransferase
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CAS REGISTRY NUMBER
COMMENTARY hide
125390-02-5
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ORGANISM
COMMENTARY hide
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
strain Heyn.
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
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-
-
Manually annotated by BRENDA team
L. cv. Vitasso
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-
Manually annotated by BRENDA team
strain N2
Uniprot
Manually annotated by BRENDA team
cultivar Goldensun
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-
Manually annotated by BRENDA team
ATCC 30929
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-
Manually annotated by BRENDA team
-
-
-
Manually annotated by BRENDA team
sacred lotus, cultivar Donghe
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-
Manually annotated by BRENDA team
cv. Bright Yellow-2
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-
Manually annotated by BRENDA team
; PCC7120
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-
Manually annotated by BRENDA team
Nostoc sp. PCC7120
PCC7120
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-
Manually annotated by BRENDA team
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SwissProt
Manually annotated by BRENDA team
isoform PCS1; birch-leaf pear
UniProt
Manually annotated by BRENDA team
Saccharomyces pombe
-
-
-
Manually annotated by BRENDA team
strain Wib.
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-
Manually annotated by BRENDA team
tomato, strain Mill. cv. VFNT-Cherry
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-
Manually annotated by BRENDA team
strain SB1969
UniProt
Manually annotated by BRENDA team
strain SB1969
UniProt
Manually annotated by BRENDA team
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
acetamido-fluorescein-glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-acetamido-fluorescein-Cys-Gly
show the reaction diagram
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-
-
-
?
benzyl-glutathione + [Glu(-Cys)]n Gly
Gly + [Glu(-Cys)]n-Glu-S-benzyl-Cys-Gly
show the reaction diagram
-
-
-
-
?
bimane-glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-bimane-Cys-Gly
show the reaction diagram
glutathione + [Glu(-Cys)]n -Gly
Gly + [Glu(-Cys)]n+1 -Gly
show the reaction diagram
glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n+1-Gly
show the reaction diagram
monobromobimane-glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-monobromobimane-Cys-Gly
show the reaction diagram
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-
-
-
?
monochlorobimane + ?
?
show the reaction diagram
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-
-
-
?
nitrobenzyl-glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-nitrobenzyl-Cys-Gly
show the reaction diagram
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-
-
-
?
S-butylglutathione + [Glu(-Cys)]n Gly
Gly + [Glu(-Cys)]n-Glu-S-butyl-Cys-Gly
show the reaction diagram
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-
-
-
?
S-ethylglutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-ethyl-Cys-Gly
show the reaction diagram
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-
-
-
?
S-hexylglutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-hexyl-Cys-Gly
show the reaction diagram
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-
-
-
?
S-methylglutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-methyl-Cys-Gly
show the reaction diagram
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n = 2,3
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-
?
S-propylglutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-propyl-Cys-Gly
show the reaction diagram
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-
-
-
?
uracil-glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n-Glu-S-uracil-Cys-Gly
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
glutathione + [Glu(-Cys)]n-Gly
Gly + [Glu(-Cys)]n+1-Gly
show the reaction diagram
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
Al3+
activation of LjPCS1; activation of LjPCS3
AsO43-
Au+
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activation
Bi3+
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activation
Ca2+
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enhanced activity, in the presence of Cd2+
KH2AsO4
activation of LjPCS3
La3+
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enhanced activity, in the presence of Cd2+
Mg2+
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activation
Ni2+
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activation
Sb3+
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activator
additional information
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
Cd2+
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the activity of PCS requires Cd and is enhanced by protein phosphorylation
GSH
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the maximal activity is attained at GSH concentrations around 10 and 15 mM when the enzyme is assayed in the presence of 0.005 and 0.01 mM total Cd2+, respectively
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.5
bimane-glutathione
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6.7 - 8.6
glutathione
1.5
monobromobimane-glutathione
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SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.09
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10 ng/ml rAtPCS1
6.6
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30 ng/ml rAtPCS1
12.6
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50 ng/ml rAtPCS1
18.4
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10 ng/ml rAtPCS1, 100 ng/ml bovine serum albumin, BSA
20.9
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1 ng/ml rAtPCS1, 10000 ng/ml bovine serum albumin, BSA
24.9
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100 ng/ml rAtPCS1
25.9
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200 ng/ml rAtPCS1
30
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10 ng/ml rAtPCS1, 10000 ng/ml bovine serum albumin, BSA
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.6
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activity assay
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
37
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activity assay
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
20 - 47
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half-maximal activity at 20°C and 47°C
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
9.4
calculated from amino acid sequence
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
SOURCE
additional information
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high expression in vascular tissues
Manually annotated by BRENDA team
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
PDB
SCOP
CATH
ORGANISM
UNIPROT
Nostoc sp. (strain PCC 7120 / UTEX 2576)
Nostoc sp. (strain PCC 7120 / UTEX 2576)
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
26700
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AtPCS1-N, residues 1-221, theoretical, verified by SDS-PAGE and Western Blot analysis
31400
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AtPCS1-C, residues 222-485, theoretical, verified by SDS-PAGE and Western Blot analysis
47000
Saccharomyces pombe
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SDS-PAGE
55000
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SDS-PAGE
56500
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determined by SDS-PAGE
56900
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calculated from sequence of cDNA
95000
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gel filtration
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
homotetramer
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4 * 25000, SDS-PAGE, dimer also catalytically active
POSTTRANSLATIONAL MODIFICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
phosphoprotein
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PCS1 expressed in Escherichia coli is phosphorylated at a Thr residue near its catalytic site
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
7.5
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most stable
487888
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
-20
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30% glycerol, 500 h, small activity decrease
22
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50% activity after 34 h
42
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40 min, wild-type enzyme loses about 40% of its activity, mutant enzyme DELTA1-284 loses about 90% of its initial activity, mutant enzyme DELTA1-373 loses about 60% of ist initial activity
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
at 30% glycerol,m/v, stability enhancement
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STORAGE STABILITY
ORGANISM
UNIPROT
LITERATURE
-20°C, 10 mM Tris-HCl, pH 8.0, 1 mM mercaptoethanol, 20% glycerol and 1% bovine serum albumin
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-20°C, 10 mM Tris-HCl, pH 8.0, 10 mM 2-mercaptoethanol, 0.1% NaN3
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-20°C, best storage temperature
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Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
DEAE-Toyopearl column chromatography and HiTrap SP column chromatography; recombinant enzyme
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DEAE-Toyopearl column chromatography, HiTrap SP column chromatography, and Hitrap Q sepharose column chromatography; recombinant enzyme
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Ni2+ affinity column chromatography and HisTrap HP column chromatography
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on a Ni2+ affinity column
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recombinant enzyme
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using a HisTrap FF column
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Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
; expressed in Escherichia coli BL21(DE3) cells
AtPCS2-gene expressed in Saccharomyces cerevisiae strain INVSc1; AtPCS2-gene expressed in Schizosaccharomyces pombe strain FY254, a phytochelatin synthase knockout strain
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BjPCS1 is expressed in Escherichia coli
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expressed in a Arabidopsis thaliana cad1-3 phytochelatin-deficient mutant and in Nicotiana tabacum cultivar Petit Havana
expressed in Arabidopsis thaliana via transformation with Agrobacterium tumefaciens strain EHA105
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expressed in Brassica juncea
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expressed in Escherichia coli
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expressed in Escherichia coli BL21 Rosetta (DE3) pLysS cells
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expressed in Nicotiana tabacum and Saccharomyces cerevisiae
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expressed in Nicotiana tabacum; overexpression both in wild-type and rolB-transformed Nicotiana tabacum. Increase in Cd2+ tolerance and accumulation in the overexpressing plants is directly related to the availability of reduced glutathione, while overexpression of phytochelatin synthase does not enhance long distance root-to shoot Cd2+ transport
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expressed in Saccharomyces cerevisiae
expressed in Saccharomyces cerevisiae strain BY4741; expressed in Saccharomyces cerevisiae strain BY4741
expressed in Saccharomyces cerevisiae strain BY4742
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expressed in Saccharomyces cerevisiae strain YK44 and Nicotiana tabacum strain NC89
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expressed in Schizosaccharomyces pombe strain SP27, a phytochelatin synthase knockout strain
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expression in Escherichia coli and Saccharomyces cerevisiae to enhance tolerance to toxicity of cadmium ion
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expression in Mesorhizobium huakuii subsp. rengei B3. The PCS(At) gene is expressed under the control of the nifH promoter, which regulates the nodule-specific expression of nifH gene. Expression of the PCS(At) gene in Mesorhizobium huakuii subsp. rengei B3 increases the ability of cells to bind Cd2+ approximately 9fold to 19fold
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expression in Saccharomyces cerevisiae; expression in Saccharomyces cerevisiae. Yeast cells expressing LjPCS3 show increased in vivo tolerance to Cd
fused to a C-terminal Flag epitope; heterologously expressed in Escherichia coli
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fused to a C-terminal Flag epitope; transformed into Saccharomyces cerevisiae DTY167
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heterologous expression of AtPCS1-FLAG in Saccharomyces cerevisiae
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heterologously expressed in Escherichia coli
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into the binary plasmid vector pCB302 for introducing into Agrobacterium GV3101 strain, tobacco plants are transformed by the standard leaf disc method
into the pGEM-T easy vector for sequencing, into the vector pYES2 for expression in Saccharomyces cerevisiae cells; into the pGEM-T easy vector for sequencing, into the vector pYES2 for expression in Saccharomyces cerevisiae cells; into the pGEM-T easy vector for sequencing, into the vector pYES2 for expression in Saccharomyces cerevisiae cells; into the pGEM-T easy vector for sequencing, into the vector pYES2 for expression in Saccharomyces cerevisiae cells
into the vector pET-28b for expression in Escherichia coli BL21 Rosetta DE3 pLysS cells
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overexpressed in transgenic Arabidopsis thaliana
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overexpression in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants are highly resistant to arsenic, accumulating 20-100times more biomass on 0.25 and 0.3 mM arsenate than wild-type, however, they are hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increases to 10fold higher levels in the A2::AtPCS1 plants compared with wild-type
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overexpression of AtPCS1 in transgenic Arabidopsis. Transgenic plants with a relatively high level of expression of the 35S::AtPCS1 transgene does not result in higher Cd tolerance, but rather show higher sensitivity to Cd under some conditions. Transgenic plants showing a relatively lower level of expression of the 35S::AtPCS1 transgene show increased accumulation and tolerance of Cd compared to wild-type plants
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the coding sequences of full-length PCS1, PCS-N, residues 1-221, and PCS-C, residues 222-485, are cloned into the vector pGEM-T-Easy and subsequently into pET28b for expression in Escherichia coli BL21DE3 cells
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the plasmid pYES3-AtPCS1-FLAG is used for the transformation of Saccharomyces cerevisiae cells
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transformed into Saccharomyces cerevisiae DTY167
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transformed into Saccharomyces cerevisiae strain DTY67, hypersensitive to Cd2+-stress
truncated mutant enzymes
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with the TOPO TA Cloning kit for sequencing
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
after 24 h of exposure to 0.02 mM CdSO4, CuSO4 or ZnSO4, isoform PCS1 expression increases significantly. The increases in roots are in order of 4.34fold, 0.26fold or 2.82fold after exposure to CdSO4, CuSO4 or ZnSO4, respectively. Reduced L-glutathione stimulates isoform PCS1 expression in roots, stems and leaves (1.45-2.85fold)
enzyme expression increases gradually in the leaves when given a treatment of 0.1, 0.2 or 0.5 mM CdCl2 for 4 or 8 h
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L-buthionine-sulfoximine can inhibit isoform PCS1 expression in roots, stems and leaves
the expression of the enzyme in leaves of Nelumbo nucifera is dramatically increased in response to cadmium (0.4 mM) treatment. In the root, enzyme expression initially raises slightly after exposure to Cd, but falls back after 1 h. When exposed to cadmium (0.1 mM) stress, Arabidopsis transgenic plants heterologous expressing the enzyme accumulate more cadmium when compared with wild type
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there is no significant change in transcript accumulation of PCS1 following Cd2+ treatment
up-regulated under cadmium stress conditions
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
C109A
-
mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C109S
-
mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C113A
-
mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C113S
-
mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C56S
-
mutation abolishes Cd2+ tolerance observed with wild-type enzyme, causes negligible intracellular phytochelatin accumulation
C90A
-
mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C90S
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mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C91A
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mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
C91S
-
mutant enzyme shows similar degree of Cd2+ tolerance on DTY167 cells as the wild-type equivalent
D180A
-
the mutation abolishes Cd2+ tolerance and phytocelatin synthetic activity
DELTA1-284
-
biosynthetically active in the presence of cadmium ions and supporting phytochelatin formation at a rate that is only about 5fold lower than that of full-length AtPCS1. The loss of the C-terminal region substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals
DELTA1-373
-
almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme
DELTA222-485
-
truncation mutant is fulla sufficient for phytochelatin synthesis. The fragment may be insufficient to maintain the active form of the enzyme stably in vitro
H162A
-
the mutation abolishes Cd2+ tolerance and phytocelatin synthetic activity
R183a
-
AtPCS1 mutant, Arg183 is critical to the activity of PCS; AtPCS1-N mutant, Arg183 is critical to the activity of PCS
T49A
-
AtPCS1 mutant, Thr49 is the only residue to be phosphorylated; AtPCS1-N mutant, Thr49 is the only residue to be phosphorylated
Y55A
-
AtPCS1 mutant; AtPCS1-N mutant
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
analysis
-
a HPLC method for the analysis of the activity of phytochelatin synthase is developed
biotechnology
environmental protection
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yeast cells expressing AtPCS can be used as an inexpensive sorbent for the removal of toxic arsenic