Literature summary for 2.5.1.19 extracted from

Vande Berg, B.J.; Hammer, P.E.; Chun, B.L.; Schouten, L.C.; Carr, B.; Guo, R.; Peters, C.; Hinson, T.K.; Beilinson, V.; Shekita, A.; Deter, R.; Chen, Z.; Samoylov, V.; Bryant, C.T.; Stauffer, M.E.; Eberle, T.; Moellenbeck, D.J.; Carozzi, N.B.; Koziel, M.G.; Duck, N.B.
Characterization and plant expression of a glyphosate-tolerant enolpyruvylshikimate phosphate synthase (2008), Pest Manag. Sci., 64, 340-345.

Application

Application	Comment	Organism
biotechnology	conferring glyphosate tolerance in transgenic crop plants	Bacteria

Cloned(Commentary)

Cloned (Comment)	Organism
from genomic DNA, in cosmid vector for phage packaging and transfection in Escherichia coli JM101 or in vitro transposon mutagenesis and transformation in Escherichia coli XL1 Blue MRF, in pAX306 for transformation of Escherichia coli DH5alpha and XL1 Blue MRF, in pRSF1b for inducible expression in Escherichia coli, in pSB11 shuttle vector for integration into Agrobacterium tumefaciens LBA4404 (already harbouring pSB1) and subsequent transformation of positive Agrobacterium strains in maize embryo for generation of transgenic plants	Bacteria

Cloned (Comment)

Organism

from genomic DNA, in cosmid vector for phage packaging and transfection in Escherichia coli JM101 or in vitro transposon mutagenesis and transformation in Escherichia coli XL1 Blue MRF, in pAX306 for transformation of Escherichia coli DH5alpha and XL1 Blue MRF, in pRSF1b for inducible expression in Escherichia coli, in pSB11 shuttle vector for integration into Agrobacterium tumefaciens LBA4404 (already harbouring pSB1) and subsequent transformation of positive Agrobacterium strains in maize embryo for generation of transgenic plants

Bacteria

Protein Variants

Protein Variants	Comment	Organism
DELTAN1-22	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	Bacteria

Inhibitors

Inhibitors	Comment	Organism	Structure
glyphosate	competitor with phosphoenolpyruvate binding site, AroA(1398) transformation allows Escherichia coli DH5alpha and XL1 Blue colonies to grow on up to 200 mM glyphosate (non-transformed bacteria stop growing on 5 mM inhibitor), AroA(1398) integrated maize transgenic plant lines showed 54% survival upon glyphosate treatment compared to none of control plants	Bacteria
glyphosate	plants do not survive glyphosate spray treatment (1.78 l/ha of a 480 g AE/L formulation) in the field	plant

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0113	-	phosphoenolpyruvate	expressed in Escherichia coli	Bacteria
0.0181	-	phosphoenolpyruvate	-	plant

Organism

Organism	UniProt	Comment	Textmining
Bacteria	-	selected from soil by its ability to grow in presence of the herbicide glyphosate	-
Bacteria ATX1398	-	selected from soil by its ability to grow in presence of the herbicide glyphosate	-
plant	-	maize	-

Purification (Commentary)

Purification (Comment)	Organism
from bacterial lysate by single step cobalt affinity chromatography followed by dialysis	Bacteria

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
35.9	-	Vmax	Bacteria
47.5	-	Vmax	plant

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
shikimate 3-phosphate + phosphoenolpyruvate	pH 7, start with phosphoenolpyruvate, with or without inhibitor glyphosate	Bacteria	phosphate + 5-enolpyruvylshikimate 3-phosphate	fluorescence-based coupled enzymatic assay including xanthine oxidase, purine nucleoside phosphorylase, inosine, horseradish peroxidase and Amplex Red	?
shikimate 3-phosphate + phosphoenolpyruvate	pH 7, start with phosphoenolpyruvate, with or without inhibitor glyphosate	plant	phosphate + 5-enolpyruvylshikimate 3-phosphate	fluorescence-based coupled enzymatic assay including xanthine oxidase, purine nucleoside phosphorylase, inosine, horseradish peroxidase and Amplex Red	?
shikimate 3-phosphate + phosphoenolpyruvate	pH 7, start with phosphoenolpyruvate, with or without inhibitor glyphosate	Bacteria ATX1398	phosphate + 5-enolpyruvylshikimate 3-phosphate	fluorescence-based coupled enzymatic assay including xanthine oxidase, purine nucleoside phosphorylase, inosine, horseradish peroxidase and Amplex Red	?

Synonyms

Synonyms	Comment	Organism
5-enolpyruvylshikimate-3phosphate synthase	-	Bacteria
5-enolpyruvylshikimate-3phosphate synthase	-	plant
AroA(1398)	glyphosate-tolerant gene product from strain ATX1398 that lacks protein sequence hallmarks of class II EPSP synthases	Bacteria
AroA1398	-	Bacteria
enolpyruvylshikimate phosphate synthase	-	Bacteria
EPSP synthase	-	Bacteria
maize EPSP synthase	-	plant
maize EPSPS	-	plant

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism	Structure
28.3	-	phosphoenolpyruvate	-	Bacteria
38.4	-	phosphoenolpyruvate	-	plant

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
additional information	-	glyphosate	KI/KM (phosphoenolpyruvate): 0.01	plant
additional information	-	glyphosate	KI/KM (phosphoenolpyruvate): 14	Bacteria
0.0002	-	glyphosate	-	plant
0.161	-	glyphosate	-	Bacteria