Literature summary for 2.5.1.19 extracted from

Funke, T.; Healy-Fried, M.L.; Han, H.; Alberg, D.G.; Bartlett, P.A.; Schoenbrunn, E.
Differential inhibition of class I and class II 5-enolpyruvylshikimate 3-phosphate synthases by tetrahedral reaction intermediate analogues (2007), Biochemistry, 46, 13344-13351.

Application

Application	Comment	Organism
drug development	differential inhibition of class I and class II EPSPS by tetrahedral reaction intermediate-analogues possibly due to alteration of open-close transition during catalysis and/or upon inhibitor binding but not due to energy differences during complex formation	Staphylococcus aureus
drug development	differential inhibition of class I and class II EPSPS by tetrahedral reaction intermediate-analogues possibly due to alteration of open-close transition during catalysis and/or upon inhibitor binding but not due to energy differences during complex formation	Escherichia coli
drug development	differential inhibition of class I and class II EPSPS by tetrahedral reaction intermediate-analogues possibly due to alteration of open-close transition during catalysis and/or upon inhibitor binding but not due to energy differences during complex formation	Agrobacterium sp.

Crystallization (Commentary)

Crystallization (Comment)	Organism
in presence of 10 mM inhibitor (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 (2F-TI, PDB: 2PQ9), 2F-TI-binding induces large conformational change of Glu341 side chain compared to TI-binding, crystals: space group P2(1)2(1)2(1), unit cell parameters: a: 58.2, b: 85.1, c: 87.6, beta: 90°, hanging-drop vapour-diffusion, sodium format conditions, molecular replacement using PDB: 1G6S as model	Escherichia coli
wild-type in presence of 5 mM inhibitor (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 (2F-TI, PDB: 2PQB) or (3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid (RP-TI, PDB: 2PQC), and mutant A100G in presence of 5 mM inhibitor 2F-TI (PDB: 2PQD): 2F-TI-binding induces only slight shift of Glu354 side chain but substantial conformational change of 2F-TI molecule that is independent of residue 100 but mediated by residue Thr101 and due to rigidity of active site, RP-TI-binding induces slight movement of Glu354 side chain and occurs via salt bridge with residue Arg128 but does not alter enzymes backbone compared to the shikimate 3-phosphate bound enzyme, crystals: space group P2(1), unit cell parameters: a: 62.8-63.4, b: 44.9-45.0, c: 77.1-77.6, beta: 106.1-106.3°, hanging-drop vapour-diffusion, ammonium sulfate conditions, molecular replacement using PDB: 2GG6 as model, co-crystallization with (3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid (SP-TI) not succeeded, docking of SP-TI into substrate- or inhibitor-bound structures indicates low electrostatic interactions between enzyme and inhibitor	Agrobacterium sp.

Crystallization (Comment)

Organism

in presence of 10 mM inhibitor (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 (2F-TI, PDB: 2PQ9), 2F-TI-binding induces large conformational change of Glu341 side chain compared to TI-binding, crystals: space group P2(1)2(1)2(1), unit cell parameters: a: 58.2, b: 85.1, c: 87.6, beta: 90°, hanging-drop vapour-diffusion, sodium format conditions, molecular replacement using PDB: 1G6S as model

Escherichia coli

wild-type in presence of 5 mM inhibitor (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 (2F-TI, PDB: 2PQB) or (3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid (RP-TI, PDB: 2PQC), and mutant A100G in presence of 5 mM inhibitor 2F-TI (PDB: 2PQD): 2F-TI-binding induces only slight shift of Glu354 side chain but substantial conformational change of 2F-TI molecule that is independent of residue 100 but mediated by residue Thr101 and due to rigidity of active site, RP-TI-binding induces slight movement of Glu354 side chain and occurs via salt bridge with residue Arg128 but does not alter enzymes backbone compared to the shikimate 3-phosphate bound enzyme, crystals: space group P2(1), unit cell parameters: a: 62.8-63.4, b: 44.9-45.0, c: 77.1-77.6, beta: 106.1-106.3°, hanging-drop vapour-diffusion, ammonium sulfate conditions, molecular replacement using PDB: 2GG6 as model, co-crystallization with (3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid (SP-TI) not succeeded, docking of SP-TI into substrate- or inhibitor-bound structures indicates low electrostatic interactions between enzyme and inhibitor

Agrobacterium sp.

Protein Variants

Protein Variants	Comment	Organism
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	Agrobacterium sp.

Inhibitors

Inhibitors	Comment	Organism
(3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	RP-TI, (R)-phosphonate analogue of the (S)-tetrahedral reaction intermediate, competitive inhibitor of EPSPS forward reaction, 460fold decrease in potency compared to glyphosate-sensitive class I EPSPS from Escherichia coli	Agrobacterium sp.
(3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	RP-TI, (R)-phosphonate analogue of the (S)-tetrahedral reaction intermediate, very potent competitive inhibitor of EPSPS forward reaction, binding induces substantial conformational change in enzymes backbone and active site (e.g., Glu341 and Arg124)	Escherichia coli
(3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	RP-TI, (R)-phosphonate analogue of the (S)-tetrahedral reaction intermediate, competitive inhibitor of EPSPS forward reaction, 120-fold decrease in potency compared to glyphosate-sensitive class I EPSPS from Escherichia coli	Staphylococcus aureus
(3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	SP-TI, (S)-phosphonate analogue of the (S)-tetrahedral reaction intermediate, non-potent competitive inhibitor of EPSPS forward reaction	Agrobacterium sp.
(3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	SP-TI, (S)-phosphonate analogue of the (S)-tetrahedral reaction intermediate, moderate competitive inhibitor of EPSPS forward reaction	Escherichia coli
(3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	SP-TI, (S)-phosphonate analogue of the (S)-tetrahedral reaction intermediate, competitive inhibitor of EPSPS forward reaction	Staphylococcus aureus
(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	2F-TI, (R)-difluoromethyl analogue of the (S)-tetrahedral reaction intermediate, competitive inhibitor of EPSPS forward reaction, 8fold decrease in potency compared to glyphosate-sensitive class I EPSPS from Escherichia coli	Agrobacterium sp.
(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	2F-TI, (R)-difluoromethyl analogue of the (S)-tetrahedral reaction intermediate, very potent competitive inhibitor of EPSPS forward reaction	Escherichia coli
(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	2F-TI, (R)-difluoromethyl analogue of the (S)-tetrahedral reaction intermediate, competitive inhibitor of EPSPS forward reaction, 4-fold decrease in potency compared to glyphosate-sensitive class I EPSPS from Escherichia coli	Staphylococcus aureus
glyphosate	-	Agrobacterium sp.
glyphosate	-	Escherichia coli
glyphosate	-	Staphylococcus aureus

Organism

Organism	UniProt	Comment	Textmining
Agrobacterium sp.	Q9R4E4	-	-
Agrobacterium sp. CP4	Q9R4E4	-	-
Escherichia coli	P0A6D3	-	-
Staphylococcus aureus	-	-	-

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
shikimate 3-phosphate + phosphoenolpyruvate	via formation of a 2-(S)-tetrahedral reaction intermediate (TI), pH 7.5, 25°C, 20 min	Staphylococcus aureus	phosphate + 5-enpolpyruvylshikimate 3-phosphate	examination by malachite green phosphate-release assay	?
shikimate 3-phosphate + phosphoenolpyruvate	via formation of a 2-(S)-tetrahedral reaction intermediate (TI), pH 7.5, 25°C, 30 min	Escherichia coli	phosphate + 5-enpolpyruvylshikimate 3-phosphate	examination by malachite green phosphate-release assay	?
shikimate 3-phosphate + phosphoenolpyruvate	via formation of a 2-(S)-tetrahedral reaction intermediate (TI), pH 7.5, 25°C, 30 min	Agrobacterium sp.	phosphate + 5-enpolpyruvylshikimate 3-phosphate	examination by malachite green phosphate-release assay	?
shikimate 3-phosphate + phosphoenolpyruvate	via formation of a 2-(S)-tetrahedral reaction intermediate (TI), pH 7.5, 25°C, 30 min	Agrobacterium sp. CP4	phosphate + 5-enpolpyruvylshikimate 3-phosphate	examination by malachite green phosphate-release assay	?

Synonyms

Synonyms	Comment	Organism
5-enolpyruvylshikimate 3-phosphate synthase	class I, glyphosate-sensitive	Escherichia coli
5-enolpyruvylshikimate 3-phosphate synthase	class II, glyphosate-tolerant	Staphylococcus aureus
5-enolpyruvylshikimate 3-phosphate synthase	class II, glyphosate-tolerant	Agrobacterium sp.
EPSP synthase	-	Staphylococcus aureus
EPSP synthase	-	Escherichia coli
EPSP synthase	-	Agrobacterium sp.
EPSPS	-	Staphylococcus aureus
EPSPS	-	Escherichia coli
EPSPS	-	Agrobacterium sp.

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
0.0000039	-	(3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	+/-0.6 nM	Escherichia coli
0.0000078	-	(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	+/-0.5 nM	Escherichia coli
0.00003	-	(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	+/-6 nM	Staphylococcus aureus
0.000063	-	(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	+/-15 nM	Agrobacterium sp.
0.00045	-	(3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	+/-70 nM	Staphylococcus aureus
0.00076	-	(3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	+/-200 nM	Escherichia coli
0.0018	-	(3R,4S,5R)-4-hydroxy-5-[(2R)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	+/-0.0006 mM	Agrobacterium sp.
0.011	-	(3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	+/-0.002 mM	Staphylococcus aureus
0.076	-	(3R,4S,5R)-4-hydroxy-5-[(2S)-1-hydroxy-1-oxo-2-phosphono-propan-2-yl]oxy-3-phosphonooxy-cyclohexene-1-carboxylic acid	+/-0.009 mM	Agrobacterium sp.