The structure and domain organization ofEscherichia coliisocitrate lyase

Abstract

Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1 A resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle.

Amino acid residues involved in cold adaptation of isocitrate lyase from a psychrophilic bacterium, Colwellia maris

“…The molecular modelling of CmICL revealed that the Gln207 of CmICL is present in the barrel fold, and its side chain is directed toward the inner space of the barrel (Fig. 7), in the same way as the His184 of EcICL (Britton et al, 2000(Britton et al, , 2001Sharma et al, 2000). It has been reported that four EcICL mutants, in which His184 was replaced by Leu, Lys, Arg or Gln, are not able to form the tertiary structure, and that only the H184Q mutant shows activity (0?28 % of the wild-type EcICL activity; Diehl & McFadden, 1994).…”

“…1a). Lys194 of EcICL should be essential for the catalytic function, because an 'active-site loop' including this residue moves flexibly when the enzyme binds to the substrate and releases the reaction products (Britton et al, 2000(Britton et al, , 2001Sharma et al, 2000;cf. Fig.…”

“…EcICL has been studied more extensively than its counterparts in other organisms with regard to the catalytic mechanism and crystal structure (Britton et al, 2001;Ko & McFadden, 1990;Ko et al, 1991Ko et al, , 1992Diehl & McFadden, 1993Rehman & McFadden, 1996, 1997a. We previously reported that, in the cold-adapted CmICL, several amino acid residues strictly conserved in the ICLs of many organisms, including E. coli, are substituted, and some unique insertions of 3-36 amino acid residues are present (Watanabe et al, 2002a).…”

“…We previously reported that the ICLs of two psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea, are homotetrameric, as are their counterparts in other organisms, including Escherichia coli, that they are typical cold-adapted enzymes, and that the expression of the genes encoding these enzymes is cold-inducible (Watanabe et al, 2001(Watanabe et al, , 2002a(Watanabe et al, , 2002b. Furthermore, the recently resolved crystal structures of ICLs and their complexes with ligands from several organisms can provide us with useful information to understand their catalytic function (Britton et al, 2000(Britton et al, , 2001Sharma et al, 2000). We previously found that about 20 amino acid residues among those strictly conserved in ICLs from various organisms are substituted in the corresponding enzyme of C. maris (CmICL) (Watanabe et al, 2002a).…”

Structure and Function of PA4872 from Pseudomonas aeruginosa, a Novel Class of Oxaloacetate Decarboxylase from the PEP Mutase/Isocitrate Lyase Superfamily,

“…All of these enzymes catalyze the transformation of an α-oxocarboxylate substrate via an oxyanion intermediate and/or transition state (Figure 1) with the aid of a Mg 2+ cofactor. Structure determinations have been carried out for each of these enzymes, with the exception of carboxyPEP mutase and oxaloacetate hydrolase, to define a conserved catalytic scaffold located at the C-terminal edge of the main-frame α/β-barrel (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11). The catalytic scaffold provides a binding pocket for the α-oxocarboxylate substrate that includes residues engaged in hydrogen bond formation with the carboxylate substituent, and residues that bind the Mg 2+ cofactor directly or via water molecules.…”