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.
Subject(s)
Escherichia coli/enzymology , Isocitrate Lyase/chemistry , Alanine/genetics , Amino Acid Sequence , Amino Acid Substitution , Aspergillus nidulans/enzymology , Binding Sites , Catalysis , Crystallography, X-Ray , Cysteine/genetics , Isocitrate Lyase/metabolism , Models, Molecular , Molecular Sequence Data , Phosphopyruvate Hydratase/chemistry , Protein Conformation , Protein Folding , Protein Structure, Quaternary , Protein Structure, Tertiary , Sequence Homology, Amino AcidABSTRACT
The NADP-dependent beta-keto acyl carrier protein reductase (BKR) from E. coli has been crystallized by the hanging-drop method of vapour diffusion using poly(ethylene glycol) of average molecular weight 1450. The crystals belong to the hexagonal space group P6122 or P6522 with unit-cell dimensions a = b = 67.8, c = 355.8 A. Calculated values for Vm and consideration of the packing suggest that the asymmetric unit contains a dimer. BKR catalyses the first reductive step in the elongation cycle of fatty-acid biosynthesis. It shares extensive sequence homology with the enzyme which catalyzes the second reductive step in the cycle, enoyl acyl carrier protein reductase (ENR), and thus provides an opportunity to study the evolution of enzyme function in a metabolic pathway. The structure determination will permit the analysis of the molecular basis of its catalytic mechanism and substrate specificity.
Subject(s)
Alcohol Oxidoreductases/chemistry , Escherichia coli/enzymology , 3-Oxoacyl-(Acyl-Carrier-Protein) Reductase , Crystallization , Diffusion , Software , X-Ray DiffractionABSTRACT
Isocitrate lyase (ICL) from the filamentous fungus Aspergillus nidulans catalyzes the first committed step of the carbon-conserving glyoxylate bypass. This enzyme has been crystallized by the hanging-drop method of vapour diffusion using polyethylene glycol 2000 as the precipitant. Diffraction patterns show that the crystals diffract to beyond 2.5 A and are probably in space group P4(2)2(1)2 with unit-cell dimensions of a = b = 91.9 and c = 152.7 A, with one molecule in the asymmetric unit. The elucidation of the structure of this enzyme to high resolution will advance the understanding of how the metabolic branch point between the tricarboxylic acid cycle and the glyoxylate bypass is controlled by the affinity of ICL for its substrate isocitrate and contribute to a programme of rational drug design.
