1OWC

Three Dimensional Structure Analysis Of The R109L Variant of the Type II Citrate Synthase From E. Coli

Summary for 1OWC

• Entry DOI: 10.2210/pdb1owc/pdb
• Related: 1K3P 1OWB
• Descriptor: Citrate synthase, SULFATE ION (3 entities in total)
• Functional Keywords: allostery, nadh, type ii citrate synthase, e. coli, r109l, transferase
• Biological source: Escherichia coli
• Total number of polymer chains: 2
• Total formula weight: 96738.29

Authors

Stokell, D.J.,Donald, L.J.,Maurus, R.,Nguyen, N.T.,Sadler, G.,Choudhary, K.,Hultin, P.G.,Brayer, G.D.,Duckworth, H.W. (deposition date: 2003-03-28, release date: 2004-05-18, Last modification date: 2023-08-16)

Primary citation

Stokell, D.J.,Donald, L.J.,Maurus, R.,Nguyen, N.T.,Sadler, G.,Choudhary, K.,Hultin, P.G.,Brayer, G.D.,Duckworth, H.W.
Probing the roles of key residues in the unique regulatory NADH binding site of type II citrate synthase of Escherichia coli.
J.Biol.Chem., 278:35435-35443, 2003

PubMed Abstract: The citrate synthase of Escherichia coli is an example of a Type II citrate synthase, a hexamer that is subject to allosteric inhibition by NADH. In previous crystallographic work, we defined the NADH binding sites, identifying nine amino acids whose side chains were proposed to make hydrogen bonds with the NADH molecule. Here, we describe the functional properties of nine sequence variants, in which these have been replaced by nonbonding residues. All of the variants show some changes in NADH binding and inhibition and small but significant changes in kinetic parameters for catalysis. In three cases, Y145A, R163L, and K167A, NADH inhibition has become extremely weak. We have used nanospray/time-of-flight mass spectrometry, under non-denaturing conditions, to show that two of these, R163L and K167A, do not form hexamers in response to NADH binding, unlike the wild type enzyme. One variant, R109L, shows tighter NADH binding. We have crystallized this variant and determined its structure, with and without bound NADH. Unexpectedly, the greatest structural changes in the R109L variant are in two regions outside the NADH binding site, both of which, in wild type citrate synthase, have unusually high mobilities as measured by crystallographic thermal factors. In the R109L variant, both regions (residues 260 -311 and 316-342) are much less mobile and have rearranged significantly. We argue that these two regions are elements in the path of communication between the NADH binding sites and the active sites and are centrally involved in the regulatory conformational change in E. coli citrate synthase.

PubMed: 12824188
DOI: 10.1074/jbc.M302786200

Experimental method

X-RAY DIFFRACTION (2.2 Å)