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G100A
-
the mutant has a 30fold increase in the IC50 value for glyphosate and slightly reduced specific activity compared to the wild type enzyme
L105P
-
the mutant shows 20% catalytic activity compared to wild type enzyme
G100A
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the mutant has a 30fold increase in the IC50 value for glyphosate and slightly reduced specific activity compared to the wild type enzyme
-
L105P
-
the mutant shows 20% catalytic activity compared to wild type enzyme
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agriculture
-
glyphosate resistance is correlated with the degree of enzyme over-expression in plants
H385L
-
0.2% of wild-type activity, 2fold activation at 100 mM NH4Cl
H385N
-
6% of wild-type activity
P105S
-
69% of wild-type activity, 8fold activation at 100 mM NH4Cl
R24D
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0.8% of wild-type activity, 2fold activation at 100 mM NH4Cl
E145G/N163H/N267S/P318R/M377V/M425T/P438L
mutant is able to restore the growth of the Escherichia coli mutant ER2799, with the 5-enolpyruvylshikimate-3-phosphate synthase gene deleted from its genome, on an M9 minimal medium containing 300 mM glyphosate. The effectiveness of amino acid alterations N267S, P318R, and M245T on glyphosate tolerance is in the decreasing order P318R, M425T, N267S
M425T
mutation identified in a multiple mutant enzyme resistant to glyphosate
N267S
mutation identified in a multiple mutant enzyme resistant to glyphosate
N267S/P318R/M425T
mutant enzyme shows high tolerance against glyphosate
P318R
mutation identified in a multiple mutant enzyme resistant to glyphosate
P381L
-
similiar glyphosate sensitivity like wild-type
T102I/P106S
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the mutant shows very high-level (2647fold) in vitro resistance to glyphosate relative to the wild type enzyme
A183T
38fold decrease in phosphoenolpyruvate-binding affinity, more solvent-exposed tryptophan residues and lower stability against guanidine hydrochloride compared to wild-type and mutant G96A, midpoint guanidine hydrochloride concentration of unfolding: 0.7 M, higher structural flexibility and decrease of secondary structure (28% alpha helix, 35% beta sheet) compared to wild-type (40% alpha helix, 31% beta sheet) and lowest resistance against proteolysis, residue A183 located on the exterior in the N-terminal domain
D242A
-
site-directed mutagenesis, the mutation is responsible for the high increase in activity
D313C
compared to wild-type mutations of D313 causes kcat to decrease. In the mutant D313C the kcat is smaller than in other mutants (1200fold). Cys is ionizable and can potentially act as an acid/base catalyst, or the thiolate form can stabilize cationic intermediates electrostatically. This accounts for the higher catalytic activity for D313C than for D313A: The effects on Km(3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest
D313L
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
D348A
-
site-directed mutagenesis, the mutation is responsible for the high increase in activity
D49A
41% of wild-type activity
E341C
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
E341M
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
K340A
2.4% of wild-type activity
K411A
10.4% of wild-type activity
N94A
50% of wild-type activity
P101A
slight decrease in catalytic efficiency, decreased inhibitory potency of glyphosate
P101G
slight decrease in catalytic efficiency, decreased inhibitory potency of glyphosate
P101L
lowest catalytic efficiency, decreased inhibitory potency of glyphosate due to long-range conformational changes
Q171A
1.7% of wild-type activity
R100A
-
site-directed mutagenesis, the mutation is responsible for the high increase in activity
R100M
0.2% of wild-type activity
R124A
19.6% of wild-type activity
R27A
binding of shikimate 3-phosphate is abolished
R344K
31.7% of wild-type activity
R344M
16.3% of wild-type activity
R386M
15.8% of wild-type activity
T42M
-
site-directed mutagenesis, the mutation is responsible for the glyphosate resistance and the low Km for phosphoenolpyruvate
T971I
the single site T97I mutation renders the enzyme sensitive to glyphosate and causes a substantial decrease in the affinity for phosphoenolpyruvate. Km (3-phosphoshikimate): 0.077 mM, Km (phosphoenolpyruvate): 0.38, kcat/Km (phosphoenolpyruvate): 23000/Msec, kcat/Km (3-phosphoshikimate): 1200000/Msec
T97I/P101S
mutant is essentially insensitive to glyphosate (Ki 2.4 mM) but maintains high affinity for the substrate phosphoenolpyruvate (Km: 0.1 mM) and 3-phosphoshikimate (Km: 0.077 mM). kcat/Km (phosphoenolpyruvate): 57000/Msec, kcat/Km (3-phosphoshikimate): 740000/Msec. The crystal structure at 1.7 A resolution reveals that the dual mutation causes a shift of residue Gly96 toward the glyphosate binding site, impairing efficient binding of glyphosate, while the side chain of Ile97 points away from the substrate binding site, facilitating phosphoenolpyruvate utilization
Y200F
1% of wild-type activity
agriculture
the enzyme is efficiently utilized to develop transgenic glyphosate-tolerant crops
G96A
-
glyphosate-insensitive
A187T
-
the mutation alters glyphosate resistance and slightly reduces enzyme activity
T101A
-
the mutation alters glyphosate resistance and strongly reduces enzyme activity
T101A/A187T
-
the mutation alters glyphosate resistance and reduces enzyme activity
agriculture
-
genetically engineered rice endogenous enzyme gene overexpressing 5-enolpyruvoylshikimate-3-phosphate synthase can increase glyphosate herbicide-resistance of cultivated rice. This type of enzyme transgene can enhance the fecundity of rice crop-weed hybrid offspring in the absence of glyphosate, stimulating great concerns over undesired environmental impacts of transgene flow to populations of wild relatives
P106L
P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. 3-fold increase in glyphosate resistance of Escherichia coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate is decreased about 70-fold and 4.6-fold, respectively, compared to wild-type
G97A/T98I/P102S
-
the mutations significantly improve glyphosate resistance
A242V
-
the mutation leads to sensitivity to 8.768% glyphosate
A329T
-
the mutation leads to sensitivity to 8.768% glyphosate
G292C
-
the mutation leads to sensitivity to 8.768% glyphosate
G292S
-
the mutation does not affect sensitivity to glyphosate
L184F
-
the mutation leads to sensitivity to 8.768% glyphosate
L35F
-
the mutation leads to sensitivity to 8.768% glyphosate
N265S
-
the mutation leads to sensitivity to 8.768% glyphosate
P71L
-
the mutation leads to sensitivity to 8.768% glyphosate
R21C
-
the mutation leads to sensitivity to 8.768% glyphosate
T258A
-
the mutation leads to sensitivity to 8.768% glyphosate
A329T
-
the mutation leads to sensitivity to 8.768% glyphosate
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G292S
-
the mutation does not affect sensitivity to glyphosate
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N265S
-
the mutation leads to sensitivity to 8.768% glyphosate
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R21C
-
the mutation leads to sensitivity to 8.768% glyphosate
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T258A
-
the mutation leads to sensitivity to 8.768% glyphosate
-
A329T
-
the mutation leads to sensitivity to 8.768% glyphosate
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G292S
-
the mutation does not affect sensitivity to glyphosate
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N265S
-
the mutation leads to sensitivity to 8.768% glyphosate
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R21C
-
the mutation leads to sensitivity to 8.768% glyphosate
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T258A
-
the mutation leads to sensitivity to 8.768% glyphosate
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M129W
no enzymatic activity
N130D
in a growth complementation assay no difference to wild-type
N130G
in a growth complementation assay no difference to wild-type
N130H
in a growth complementation assay no difference to wild-type
N130L
in a growth complementation assay no difference to wild-type
N130P
partial impaiment in growth complementation assay
N130W
partial impaiment in growth complementation assay, mutant shows a significant decrease in resistance to glyphosate, Km (phosphoenolpyruvate): 0.025 mM
P128R
no enzymatic activity
R127E
no enzymatic activity
R127K
no enzymatic activity
R131E
no enzymatic activity
R131K
no enzymatic activity
N130S
-
glyphosate resistance enhanced to 200 mM and glyphosate affinity reduced (2.5fold increase in IC50 and 2fold increase in Ki for glyphosate compared to wild-type) but similar catalytic activity as wild-type, generated by error-prone PCR
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G96A
mutant with improved resistance towards glyphosate
T38K/R40V/R222Q/S224V/I225V/Q226K
mutant with the c-strand of subdomain 3 and the f-strand of subdomain 5 substituted by the corresponding region of the Escherichia coli enzyme. The mutant enzyme exhibits greater sensitivity to glyphosate than the wild type with little change of affinity for substrates shikimate-3-phosphate and phosphoenolpyruvate
G96A
-
mutant with improved resistance towards glyphosate
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T38K/R40V/R222Q/S224V/I225V/Q226K
-
mutant with the c-strand of subdomain 3 and the f-strand of subdomain 5 substituted by the corresponding region of the Escherichia coli enzyme. The mutant enzyme exhibits greater sensitivity to glyphosate than the wild type with little change of affinity for substrates shikimate-3-phosphate and phosphoenolpyruvate
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T42M
-
site-directed mutagenesis, the mutation is responsible for the glyphosate resistance and the low Km for phosphoenolpyruvate
food industry
-
the enzyme can be used for cultivation of glyphosate-tolerant plants
T103I/P107S
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double mutant (47 kDa) which is widely used by genetic engineering to confer glyphosate tolerant properties, to various crops such as corn, cotton, canola, and soybean is assessed in terms of safety aspects: The expressed protein is innocuous. The double mutant enzyme does not possess any of the properties associated with known toxins or allergens, a rapid degradation in simulated gastric and intestinal fluids, and no adverse effects in mice after intravenousor oral administration is shown
A100G
glyphosate-sensitive, compared to wild-type similar inhibition constants for (3R,4S,5R)-5-[(2R)-1,1-difluoro-3-hydroxy-3-oxo-2-phosphonooxy-propan-2-yl]oxy-4-hydroxy-3-phosphonooxy-cyclohexene-1-carboxylic acid, (3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid, and (3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid
A100G
-
glyphosate-sensitive, compared to wild-type similar inhibition constants for (3R,4S,5R)-5-[(2R)-1,1-difluoro-3-hydroxy-3-oxo-2-phosphonooxy-propan-2-yl]oxy-4-hydroxy-3-phosphonooxy-cyclohexene-1-carboxylic acid, (3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid, and (3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid
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DELTAN1-22
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lacking N-terminal 22 residues including critical amino acid Lys20, fails to provide glyphosate tolerance compared to full-length enzyme when transformed in glyphosate-sensitive Escherichia coli DH5alpha and XL1 Blue
DELTAN1-22
Bacteria ATX1398
-
lacking N-terminal 22 residues including critical amino acid Lys20, fails to provide glyphosate tolerance compared to full-length enzyme when transformed in glyphosate-sensitive Escherichia coli DH5alpha and XL1 Blue
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P106S
-
glyphosate-insensitive
P106S
-
the mutant shows high in vitro resistance to glyphosate relative to the wild type enzyme
D313A
0.42% of wild-type activity
D313A
site-directed mutagenesis, comparison of the mutant active site and substrate binding structures to those of the wild-type enzyme
D313A
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
D313N
5% of wild-type activity
D313N
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
E341A
0.3% of wild-type activity
E341A
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
E341Q
10% of wild-type activity
E341Q
compared to wild-type mutations of D313 causes kcat to decrease up to 30000fold while the effects on Km (3-phosphoshikimate) or Km (phosphoenolpyruvate) are modest, never more than 40fold
G96A
-
glyphosate-insensitive
G96A
glyphosate-insensitive, 31fold decrease in phosphoenolpyruvate-binding affinity, little change with respect to structural flexibility and stability compared to wild-type, lower structural flexibility than wild-type and mutants A183T and G96A/A183T, midpoint guanidine hydrochloride concentration of unfolding: 1.1 M, residue Gly96 located at interphase between two domains
G96A
the mutation confers resistance to glyphosate
G96A/A183T
-
mutant enzyme is resistant to glyphosate
G96A/A183T
glyphosate-insensitive, 8fold decrease in phosphoenolpyruvate-binding affinity, more solvent-exposed tryptophan residues and lower stability against guanidine hydrochloride compared to wild-type and mutant G96A, midpoint guanidine hydrochloride concentration of unfolding: 0.65 M, higher structural flexibility and decrease of secondary structure (36% alpha helix, 38% beta sheet) compared to wild-type (40% alpha helix, 31% beta sheet)
H385A
0.08% of wild-type activity
H385A
compared to wild-type mutation of H385 causes a 78fold decrease in kcat. Mutation has a 37fold effect on Km (phosphoenolpyruvate), almost no effect on Km (3-phosphoshikimate)
K22A
0.7% of wild-type activity
K22A
compared to wild-type, K22A mutation causes a more than760fold increase in Km (phosphoenolpyruvate) and a more than 100fold increase in Km(3-phosphoshikimate), indicating an important role in binding both substrates
K22R
3% of wild-type activity
K22R
compared to wild-type, K22R mutation has only a 10fold effect on kcat, presumably reflecting electrostatic stabilization of the anionic leaving groups in tetrahedral intermediate breakdown or possibly general acid catalysis by the guanidinium group. K22A mutation causes a 60fold increase in Km (phosphoenolpyruvate)
P101S
slight decrease in catalytic efficiency, decreased inhibitory potency of glyphosate due to long-range conformational changes, flexibility of Ser101 side chain disables hydrophobic bonding with carbonyl oxygen of Thr97
P101S
the substrate binding affinities, as reflected by the respective Km values, are only slightly decreased for the P101S mutant. Km (3-phosphoshikimate): 0.071 mM, Km (phosphoenolpyruvate): 0.071, kcat/Km (phosphoenolpyruvate): 230000/Msec, kcat/Km (3-phosphoshikimate): 240000/Msec. Mutant P101S is moderately inhibited by glyphosate
T355S
mutation results in a catalytically more efficient glyphosate-resistant enzyme
T355S
-
mutation results in a catalytically more efficient glyphosate-resistant enzyme
-
G99A
-
KM-value for 5-enolpyruvylshikimate 3-phosphate is 86% of the wild-type value, KM-value for phosphate is 1.3fold higher than wild-type value. Turnover-number for 5-enolpyruvylshikimate 3-phosphate is 6.2fold higher than wild-type value, turnover-number for phosphate is 5.4fold lower than wild-type value
G99A
-
similar substrate affinities and decreased turnover compared to wild-type, fails to complement aroA auxotrophy in transformed Escherichia coli
L104P
-
KM-value for 5-enolpyruvylshikimate 3-phosphate is 1.14fold higher than the wild-type value, KM-value for phosphate is 4.4fold higher than wild-type value. Turnover-number for 5-enolpyruvylshikimate 3-phosphate is 2.7fold lower than wild-type value, turnover-number for phosphate is 1.9fold lower than wild-type value
L104P
-
lower affinity to phosphate and decreased turnover compared to wild-type, fails to complement aroA auxotrophy in transformed Escherichia coli
N130S
-
glyphosate resistance enhanced to 200 mM and glyphosate affinity reduced (2.5fold increase in IC50 and 2fold increase in Ki for glyphosate compared to wild-type) but similar catalytic activity as wild-type, generated by error-prone PCR
N130S
in a growth complementation assay no difference to wild-type, Km (phosphoenolpyruvate): 0.032 mM
additional information
-
construction of chimeric mutants by fusing the N-terminal part of the Escherichia coli enzyme with the C-terminal part of the enzyme from Salmonella typhimurium, the chimeric mutant shows a 6fold lower Km for phosphoenolpyruvate, a 5fold higher Ki for glyphosate, and a 9fold higher specific activity than the wild-type enzyme from Escherichia coli
additional information
-
construction of chimeric mutants by fusing the N-terminal part of the Escherichia coli enzyme with the C-terminal part of the enzyme from Salmonella typhimurium, the chimeric mutant shows a 6fold lower Km for phosphoenolpyruvate, a 5fold higher Ki for glyphosate, and a 9fold higher specific activity than the wild-type enzyme from Escherichia coli, construction of glyphosate-resistant transgenic Nicotiana tabacum plants expressing the chimeric mutant aroA-M1 using the Agrobacterium tumefaciens strain LBA 4404 infection system
additional information
-
construction of chimeric mutants by fusing the N-terminal part of the Escherichia coli enzyme with the C-terminal part of the enzyme from Salmonella typhimurium, i.e. mutant construct aroA-M1, the chimeric mutant shows a 6fold lower Km for phosphoenolpyruvate, a 5fold higher Ki for glyphosate, and a 9fold higher specific activity than the wild-type enzyme from Escherichia coli, construction of glyphosate-resistant transgenic Nicotiana tabacum plants expressing the chimeric mutant aroA-M1 using the Agrobacterium tumefaciens strain LBA 4404 infection system
additional information
-
construction of chimeric mutants by fusing the N-terminal part of the Escherichia coli enzyme with the C-terminal part of the enzyme from Salmonella typhimurium, the chimeric mutant shows a 6fold lower Km for phosphoenolpyruvate, a 5fold higher Ki for glyphosate, and a 9fold higher specific activity than the wild-type enzyme from Escherichia coli