Literature summary for 2.5.1.54 extracted from

Cross, P.J.; Pietersma, A.L.; Allison, T.M.; Wilson-Coutts, S.M.; Cochrane, F.C.; Parker, E.J.
Neisseria meningitidis expresses a single 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase that is inhibited primarily by phenylalanine (2013), Protein Sci., 22, 1087-1099.

Cloned(Commentary)

Cloned (Comment)	Organism
gene aroG, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21 (DE3)	Neisseria meningitidis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified enzyme mutant S213G with phosphoenolpyruvate and Mn2+, hanging drop vapor diffusion, mixing of 0.001 ml of 11 mg/mL in 10 mM BTP buffer, pH 7.3, in a 1:1 v/v with a reservoir solution containing 0.2 M trimethylamine N-oxide, 0.1 M Tris, pH 8.5, 15-20% w/v PEG 2000 MME, 0.4 mM MnSO4, and 0.4 mM phosphoenolpyruvate, equilibration against 0.5 ml of reservoir solution, 20°C, 3 days, X-ray diffractin structure determination and analysis at 2.0-2.1 A resolution	Neisseria meningitidis

Crystallization (Comment)

Organism

purified enzyme mutant S213G with phosphoenolpyruvate and Mn2+, hanging drop vapor diffusion, mixing of 0.001 ml of 11 mg/mL in 10 mM BTP buffer, pH 7.3, in a 1:1 v/v with a reservoir solution containing 0.2 M trimethylamine N-oxide, 0.1 M Tris, pH 8.5, 15-20% w/v PEG 2000 MME, 0.4 mM MnSO4, and 0.4 mM phosphoenolpyruvate, equilibration against 0.5 ml of reservoir solution, 20°C, 3 days, X-ray diffractin structure determination and analysis at 2.0-2.1 A resolution

Neisseria meningitidis

Protein Variants

Protein Variants	Comment	Organism
S213G	site-directed mutagenesis, a single Ser residue at the bottom of the inhibitor-binding cavity is substituted to Gly, which alters inhibitor specificity from L-Phe to L-Tyr	Neisseria meningitidis

Inhibitors

Inhibitors	Comment	Organism
L-Phe	strong feedback inhibition	Neisseria meningitidis
L-Trp	feedback inhibition	Neisseria meningitidis
L-Tyr	feedback inhibition	Neisseria meningitidis
additional information	undergoes no significant conformational change on inhibitor binding, crystal structure analysis, overview. Allosteric response arising from changes in protein motion rather than conformation, and suggest ligands that modulate protein dynamics may be effective inhibitors of this enzyme	Neisseria meningitidis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
additional information	-	additional information	Michaelis-Menten kinetics, kinetics in presence of inhibitors L-phe and L-Tyr	Neisseria meningitidis
0.0087	-	phosphoenolpyruvate	pH 6.8, 30°C, mutant S213G	Neisseria meningitidis
0.011	-	phosphoenolpyruvate	pH 6.8, 30°C, wild-type enzyme	Neisseria meningitidis
0.04	-	D-erythrose 4-phosphate	pH 6.8, 30°C, mutant R126S	Neisseria meningitidis
0.043	-	D-erythrose 4-phosphate	pH 6.8, 30°C, wild-type enzyme	Neisseria meningitidis

Metals/Ions

Metals/Ions	Comment	Organism
Cd2+	activates	Neisseria meningitidis
Co2+	activates	Neisseria meningitidis
Cu2+	activates slightly	Neisseria meningitidis
Fe2+	activates slightly	Neisseria meningitidis
Mn2+	activates, best activating metal ion. The metal ion is coordinated by four protein ligands Cys63, His 270, Glu304, and Asp324	Neisseria meningitidis
additional information	no effect by Mg2+, Ni2+, Ca2+, Ba2+, and Sr2+	Neisseria meningitidis
Zn2+	activates	Neisseria meningitidis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
phosphoenolpyruvate + D-erythrose 4-phosphate + H2O	Neisseria meningitidis	-	3-deoxy-D-arabino-hept-2-ulosonate 7-phospate + phosphate	-	?
phosphoenolpyruvate + D-erythrose 4-phosphate + H2O	Neisseria meningitidis MC58	-	3-deoxy-D-arabino-hept-2-ulosonate 7-phospate + phosphate	-	?

Organism

Organism	UniProt	Comment	Textmining
Neisseria meningitidis	Q9K169	serotype B, gene aroG	-
Neisseria meningitidis MC58	Q9K169	serotype B, gene aroG	-
no activity in Homo sapiens	-	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain BL21 (DE3) by anion exchange chromatography, ultrafiltration, and gel filtration	Neisseria meningitidis

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
phosphoenolpyruvate + D-erythrose 4-phosphate + H2O	-	Neisseria meningitidis	3-deoxy-D-arabino-hept-2-ulosonate 7-phospate + phosphate	-	?
phosphoenolpyruvate + D-erythrose 4-phosphate + H2O	phosphoenolpyruvate interacts with conserved residues Arg167, Arg236, Lys188, Arg94, and Lys99, condensation reaction	Neisseria meningitidis	3-deoxy-D-arabino-hept-2-ulosonate 7-phospate + phosphate	-	?
phosphoenolpyruvate + D-erythrose 4-phosphate + H2O	-	Neisseria meningitidis MC58	3-deoxy-D-arabino-hept-2-ulosonate 7-phospate + phosphate	-	?
phosphoenolpyruvate + D-erythrose 4-phosphate + H2O	phosphoenolpyruvate interacts with conserved residues Arg167, Arg236, Lys188, Arg94, and Lys99, condensation reaction	Neisseria meningitidis MC58	3-deoxy-D-arabino-hept-2-ulosonate 7-phospate + phosphate	-	?

Subunits

Subunits	Comment	Organism
homotetramer	a dimer of tight dimers, crystal structure analysis	Neisseria meningitidis
More	the monomer of Nme-DAH7PS is a (beta/alpha)8 TIM-barrel	Neisseria meningitidis

Synonyms

Synonyms	Comment	Organism
3-deoxy-D-arabino-heptulosonate 7-phosphate synthase	-	Neisseria meningitidis
DAH7PS	-	Neisseria meningitidis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Neisseria meningitidis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
14.8	-	D-erythrose 4-phosphate	pH 6.8, 30°C, wild-type enzyme	Neisseria meningitidis
14.8	-	D-erythrose 4-phosphate	pH 6.8, 30°C, mutant R126S	Neisseria meningitidis
25	-	phosphoenolpyruvate	pH 6.8, 30°C, wild-type enzyme	Neisseria meningitidis
25	-	phosphoenolpyruvate	pH 6.8, 30°C, mutant S213G	Neisseria meningitidis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6.8	-	assay at	Neisseria meningitidis

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism	Structure
additional information	-	additional information	inhibition kinetics of wld-type and mutant enzymes, overview	Neisseria meningitidis

General Information

General Information	Comment	Organism
metabolism	the enzyme catalyzes the first reaction in the shikimate pathway leading to the biosynthesis of aromatic metabolites including the aromatic acids L-Trp, L-Phe, and L-Tyr. As the entry point, feedback inhibition of DAH7PS by pathway end products is a key mechanism for the control of pathway flux	Neisseria meningitidis
additional information	allosteric response arising from changes in protein motion rather than conformation, and suggest ligands that modulate protein dynamics may be effective inhibitors of this enzyme. The nature of the extra barrel extensions is directly implicated in the sensitivity of the DAH7PS enzymes to allosteric control	Neisseria meningitidis

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
370	-	D-erythrose 4-phosphate	pH 6.8, 30°C, mutant R126S	Neisseria meningitidis
590	-	D-erythrose 4-phosphate	pH 6.8, 30°C, wild-type enzyme	Neisseria meningitidis
1700	-	phosphoenolpyruvate	pH 6.8, 30°C, mutant S213G	Neisseria meningitidis
2300	-	phosphoenolpyruvate	pH 6.8, 30°C, wild-type enzyme	Neisseria meningitidis