title: Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis.
abstract: The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.
authors.affiliation: Department of Applied Biological Chemistry, School of Life and Environmental Sciences, Osaka Prefecture University, Naka-ku, Sakai, Osaka, Japan. mami@biochem.osakafu-u.ac.jp
authors.forename: Masami
authors.initials: M
authors.lastname: Nakazawa
authors.forename: Masaaki
authors.initials: M
authors.lastname: Nishimura
authors.forename: Kengo
authors.initials: K
authors.lastname: Inoue
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Ueda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Inui
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Nakano
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Miyatake
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D005056:Euglena gracilis / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D006038:Glyoxylates / Q000378:metabolism*; D007522:Isocitrate Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008292:Malate Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D006038:Glyoxylates; D015800:Protozoan Proteins; D008292:Malate Synthase; D007522:Isocitrate Lyase