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Literature summary for 2.7.2.4 extracted from

  • Kotaka, M.; Ren, J.; Lockyer, M.; Hawkins, A.R.; Stammers, D.K.
    Structures of R- and T-state Escherichia coli aspartokinase III. Mechanisms of the allosteric transition and inhibition by lysine (2006), J. Biol. Chem., 281, 31544-31552.
    View publication on PubMed
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Cloned(Commentary)

Cloned (Comment) Organism
into plasmid pET15b, N-terminal His-tagged AKIII transformed into the B834 (DE3) met- strain, untagged AKIII ligated into plasmid pET3d and transformed into Escherichia coli BL21(DE3) pLysS for protein expression Escherichia coli

Crystallization (Commentary)

Crystallization (Comment) Organism
by sitting drop vapor-diffusion method, AKIII in the inactive T-state with bound feedback allosteric inhibitor L-lysine, to 2.8 A resolution, and in the R-state with L-aspartate and ADP, to 2.5 A resolution, unusual configuration for the regulatory ACT domains, in which ACT2 is inserted into ACT1 rather than the expected tandem repeat Escherichia coli

Inhibitors

Inhibitors Comment Organism Structure
L-lysine binding of L-lysine to the regulatory ACT1 domain in R-state AKIII instigates a series of changes that release a 'latch', the beta15-alphaK loop, from the catalytic domain, which in turn undergoes large rotational rearrangements, promoting tetramer formation and completion of the transition to the T-state. L-lysine-induced allosteric transition in AKIII involves both destabilizing the R-state and stabilizing the T-state tetramer. Rearrangement of the catalytic domain blocks the ATP-binding site, which is therefore the structural basis for allosteric inhibition of AKIII by L-lysine Escherichia coli

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+
-
 Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
-
-

Purification (Commentary)

Purification (Comment) Organism
His-tagged AKIII by nickel affinity chromatography followed by gel filtration, more than 99%, untagged AKIII by gel filtration, more than 98% Escherichia coli

Storage Stability

Storage Stability Organism
-80°C, 150 mM KCl, 20 mM Tris HCl, pH 7.4 Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
ATP + L-aspartate
-
 Escherichia coli ADP + 4-phospho-L-aspartate
-
 ?

Subunits

Subunits Comment Organism
tetramer crystallography Escherichia coli

Synonyms

Synonyms Comment Organism
AKIII
-
 Escherichia coli
aspartokinase III
-
 Escherichia coli