Literature summary for 4.3.3.7 extracted from

Soares da Costa, T.P.; Muscroft-Taylor, A.C.; Dobson, R.C.; Devenish, S.R.; Jameson, G.B.; Gerrard, J.A.
How essential is the essential active-site lysine in dihydrodipicolinate synthase? (2010), Biochimie, 92, 837-845.

Cloned(Commentary)

Cloned (Comment)	Organism
DHDPS overexpressed in Escherichia coli AT997recA-, transformed with site-directed mutants based on the pBluescript plasmid pJG001	Escherichia coli

Crystallization (Commentary)

Crystallization (Comment)	Organism
by the hanging drop-vapour diffusion method, mutants K161A and K161R solved at resolutions of 2.0 and 2.1 A, respectively. They show no changes in their secondary or tertiary structures when compared to the wild-type structure. Crystal structure of mutant K161A with pyruvate bound at the active site solved at a resolution of 2.3 A, reveals a defined binding pocket for pyruvate that is thus not dependent upon lysine 161	Escherichia coli

Crystallization (Comment)

Organism

by the hanging drop-vapour diffusion method, mutants K161A and K161R solved at resolutions of 2.0 and 2.1 A, respectively. They show no changes in their secondary or tertiary structures when compared to the wild-type structure. Crystal structure of mutant K161A with pyruvate bound at the active site solved at a resolution of 2.3 A, reveals a defined binding pocket for pyruvate that is thus not dependent upon lysine 161

Escherichia coli

Protein Variants

Protein Variants	Comment	Organism
K161A	catalytically active, significant decrease in activity. Is not inactivated when incubated with pyruvate and the reducing agent sodium borohydride. Negligible heat production associated with pyruvate binding to the mutant enzyme, consistent with the lack of Schiff base formation	Escherichia coli
K161R	catalytically active, significant decrease in activity. Is not inactivated when incubated with pyruvate and the reducing agent sodium borohydride. Negligible heat production associated with pyruvate binding to the mutant enzyme, consistent with the lack of Schiff base formation	Escherichia coli

Inhibitors

Inhibitors	Comment	Organism	Structure
lysine	inhibition of wild-type DHDPS by lysine with respect to pyruvate is partial and uncompetitive, and partial non-competitive with respect to L-aspartate 4-semialdehyde. Ethanolamine, n-butylamine, 1-amino-2-propanol, 3-amino-1-propanol, iso-butylamine and Tris-HCl cannot rescue activity	Escherichia coli
Sodium borohydride	wild-type DHDPS is inactivated when incubated with pyruvate, whereas incubation with L-aspartate 4-semialdehyde has no effect	Escherichia coli

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.12	-	L-aspartate 4-semialdehyde	mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.12	-	L-aspartate 4-semialdehyde	wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.15	-	pyruvate	wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.23	-	L-aspartate 4-semialdehyde	mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.45	-	pyruvate	mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.57	-	pyruvate	mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli

Organism

Organism	UniProt	Comment	Textmining
Escherichia coli	P0A6L2	-	-

Purification (Commentary)

Purification (Comment)	Organism
wild-type and mutants, by gel filtration	Escherichia coli

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
L-aspartate 4-semialdehyde + pyruvate	condensation reaction between both substrates via the formation of a Schiff base intermediate between pyruvate and the absolutely conserved active-site lysine. Although lysine 161 is important in the wild-type DHDPS-catalysed reaction, it is not absolutely essential for catalysis	Escherichia coli	dihydrodipicolinate + 2 H2O	-	?

Synonyms

Synonyms	Comment	Organism
DHDPS	-	Escherichia coli
dihydrodipicolinate synthase	-	Escherichia coli

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.06	-	pyruvate	mutant K161A, at 30°C, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.16	-	pyruvate	mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
45	-	pyruvate	wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
0.12	-	lysine	wild-type, with pyruvate as substrate	Escherichia coli
0.14	-	lysine	mutant K161A, with pyruvate as substrate	Escherichia coli
0.14	-	lysine	mutant K161R, with L-aspartate 4-semialdehyde as substrate	Escherichia coli
0.14	-	lysine	mutant K161R, with pyruvate as substrate	Escherichia coli
0.18	-	lysine	wild-type, with L-aspartate 4-semialdehyde as substrate	Escherichia coli
0.23	-	lysine	mutant K161A, with L-aspartate 4-semialdehyde as substrate	Escherichia coli

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.13	-	pyruvate	mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.26	-	L-aspartate 4-semialdehyde	mutant K161A, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
0.28	-	pyruvate	mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
1.3	-	L-aspartate 4-semialdehyde	mutant K161R, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
300	-	pyruvate	wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli
380	-	L-aspartate 4-semialdehyde	wild-type, in 100 mM HEPES buffer, pH 8.0, 0.2 mM NADPH, 50 microg/ml DHDPR	Escherichia coli