Literature summary for 2.5.1.19 extracted from

Dun, B.Q.; Wang, X.J.; Lu, W.; Zhao, Z.L.; Hou, S.N.; Zhang, B.M.; Li, G.Y.; Evans, T.C.; Xu, M.Q.; Lin, M.
Reconstitution of glyphosate resistance from a split 5-enolpyruvyl shikimate 3-phosphate synthase gene in Escherichia coli and transgenic tobacco (2007), Appl. Environ. Microbiol., 73, 7997-8000.

Application

Application	Comment	Organism
biotechnology	strain PCC6803 DnaE intein N-terminal and C-terminal splicing domains can reconstitute EPSPS activities and glyphosate resistance in plant	Pseudomonas fluorescens

Cloned(Commentary)

Cloned (Comment)	Organism
N-terminal fragment (until Phe295) of G2 EPSPS or the complete gene in pMEB2 (N-terminal of splicing domain of of DnaE intein, strain PCC6803) and C-terminus (from Thr296) of G2 EPSPS in pKEB12 (C-terminal of splicing domain of DnaE intein, strain PCC6803) for intein-mediated protein complementation and expression in Escherichia coli strain ER2799, complete G2 EPSPS gene, N-terminal and C-terminal fragments in pBI121 for introduction into Agrobacterium sp. strain LBA4404 and transformation in Nicotiana tabacum variant NC89 for generation of transgenic plants	Pseudomonas fluorescens

Cloned (Comment)

Organism

N-terminal fragment (until Phe295) of G2 EPSPS or the complete gene in pMEB2 (N-terminal of splicing domain of of DnaE intein, strain PCC6803) and C-terminus (from Thr296) of G2 EPSPS in pKEB12 (C-terminal of splicing domain of DnaE intein, strain PCC6803) for intein-mediated protein complementation and expression in Escherichia coli strain ER2799, complete G2 EPSPS gene, N-terminal and C-terminal fragments in pBI121 for introduction into Agrobacterium sp. strain LBA4404 and transformation in Nicotiana tabacum variant NC89 for generation of transgenic plants

Pseudomonas fluorescens

Inhibitors

Inhibitors	Comment	Organism	Structure
glyphosate	N-phosphonomethylglycine	Pseudomonas fluorescens

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
0.0952	-	phosphoenolpyruvate	+/-0.00573 mM, full-length G2 EPSPS, crude lysate from overexpressing Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
0.0968	-	phosphoenolpyruvate	+/-0.01101 mM, reconstituted G2-EPSPS (by intein-mediated protein complementation), crude lysate from transformed Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens

Molecular Weight [Da]

Molecular Weight [Da]	Molecular Weight Maximum [Da]	Comment	Organism
20400	-	C-terminal fragment (from Thr296) expressed in pKEB12, Western blot	Pseudomonas fluorescens
47000	-	full-length protein, Western blot analyses	Pseudomonas fluorescens

Organism

Organism	UniProt	Comment	Textmining
Pseudomonas fluorescens	A1Z0H6	G2	-
Pseudomonas fluorescens G2	A1Z0H6	G2	-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
4.48	-	+/-0.79 micromol/min/mg, reconstituted G2-EPSPS (by intein-mediated protein complementation), mean specific activity, crude lysate from transformed Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
4.761	-	+/-0.92 micromol/min/mg, reconstituted G2-EPSPS (by intein-mediated protein complementation), maximum velocity, crude lysate from transformed Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
7.12	-	+/-0.13 micromol/min/mg, full-length G2 EPSPS, mean specific activity, crude lysate from overexpressing Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
7.24	-	+/-0.16 micromol/min/mg, full-length G2 EPSPS, maximum velocity, crude lysate from overexpressing Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
shikimate 3-phosphate + phosphoenolpyruvate	malachite green dye assay, 28°C	Pseudomonas fluorescens	phosphate + 5-enpolpyruvylshikimate 3-phosphate	-	?
shikimate 3-phosphate + phosphoenolpyruvate	malachite green dye assay, 28°C	Pseudomonas fluorescens G2	phosphate + 5-enpolpyruvylshikimate 3-phosphate	-	?

Synonyms

Synonyms	Comment	Organism
G2 5-enolpyruvyl shikimate 3-phosphate synthase	glyphosate resistant	Pseudomonas fluorescens
G2 EPSPS	-	Pseudomonas fluorescens

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
additional information	-	glyphosate	Ki/KM (phosphoenolpyruvate): 0.36, reconstituted G2-EPSPS (by intein-mediated protein complementation), crude lysate from overexpressing Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
additional information	-	glyphosate	Ki/KM (phosphoenolpyruvate): 0.52, full-length G2 EPSPS, crude lysate from overexpressing Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
0.035	-	glyphosate	+/-0.00578 mM, reconstituted G2-EPSPS (by intein-mediated protein complementation), crude lysate from transformed Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens
0.0495	-	glyphosate	+/-0.00317 mM, full-length G2 EPSPS, crude lysate from overexpressing Escherichia coli ER2799, malachite green dye assay	Pseudomonas fluorescens