Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis
Introduction
The glyoxylate cycle is a metabolic pathway which is very important for organisms to synthesize carbohydrates from C2 compounds, as first proposed by Kornberg and Krebs (1957). This cycle has two key specific enzymes; isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 2.3.3.9). ICL catalyzes the cleavage of d-isocitrate to glyoxylate and succinate, and glyoxylate formed by the ICL reaction is condensed with acetyl-CoA to produce l-malate by the action of MS. These two enzymes ensure the bypass of two of the decarboxylation steps of the tricarboxylic acid (TCA) cycle in the synthesis of succinate. Thus, the glyoxylate cycle is very important especially under carbon limiting conditions. In certain higher plants, the glyoxylate cycle has been reported to play a pivotal role in the synthesis of carbohydrates from storage lipids during seedling (Eastmond and Graham, 2001).
Isocitrate lyases have been found in a wide range of species including bacteria (Kornberg, 1966), archaea (Serrano et al., 1998), yeast (Taylor et al., 1996), fungi (Lorenz and Fink, 2001) and higher plants (Eastmond and Graham, 2001). ICLs commonly consist of four identical subunits in either prokaryotes or eukaryotes. However, the subunit molecular mass is significantly different between bacterial (approximately 47 kDa) and eukaryotic (60–64 kDa) ICLs.
On the basis of the amino acid sequence features, MSs have been divided into two major families, isoforms A (MSA) and G (MSG). MSA with a molecular mass of about 65 kDa occurs in bacteria (Kornberg, 1966), yeast (Hartig et al., 1992), fungi (Lorenz and Fink, 2001) and higher plants (Eastmond and Graham, 2001), whereas MSG with around 80 kDa has been found only in bacteria. Certain bacteria, such as Escherichia coli, express both the two types of MSs (Molina et al., 1994).
Liu et al., 1997, Liu et al., 1995 found that ICL and MS are encoded by a single gene and expressed as a single bifunctional polypeptide in nematode Caenorhabditis elegans. The polypeptide, called bifunctional glyoxylate cycle protein (GCP), consists of two separate domains for ICL and MS, and the ICL domain locates in the amino-terminal side of the polypeptide. The physiological functions of the GCP during development in nematode have been well studied; however, the catalytic properties of the unique enzyme have not been reported.
It has been reported that Euglena gracilis, a unicellular protist containing chloroplasts, has both ICL and MS activities, and these enzyme activities are greatly enhanced when ethanol is fed as a sole carbon source (Inui et al., 1992). In the present paper, we have purified a bifunctional enzyme having the ICL and MS activities from E. gracilis grown on ethanol, and cloned cDNA encoding this bifunctional enzyme. We reported that the bifunctional glyoxylate enzyme found in E. gracilis (EgGCE), as well as the GCP in C. elegans, consists of two functional components, the ICL and MS domains, but that the ICL domain is found in the carboxy-terminal side in the EgGCE, in contrast to the nematode enzyme. In addition, it is also shown that the ICL reaction is activated in the presence of acetyl-CoA, a substrate of the MS reaction, in EgGCE; Vmax increases whereas Km for Mg2 +–isocitrate complex decreases in the presence of acetyl-CoA.
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Organism and culture
Euglena gracilis SM-ZK (Oda et al., 1982), a non-photosynthetic mutant derived from strain Z by treatment with streptomycin, was cultured in Cramer–Myers medium (Cramer and Myers, 1952), supplemented with ethanol at 85 mM as a sole carbon source, with aeration at 27 °C for 4 days.
Enzyme purification
All operations during the enzyme purification were conducted at 4 °C. E. gracilis cells grown on ethanol (about 10 g wet basis) were harvested, washed and suspended in buffer A (10 mM potassium phosphate buffer, pH
Purification of bifunctional glyoxylate cycle enzyme from E. gracilis
Typical purification steps of the bifunctional glyoxylate cycle enzyme (EgGCE) are summarized in Table 1. The bifunctional enzyme was purified about 20-fold over the crude extract prepared from Euglena cells grown on ethanol. In all the steps during the purification, the MS activity co-fractionated with the ICL activity, and the ratios of the MS activity versus the ICL activity remained constant.
The purified preparation of EgGCE revealed a single protein band with a molecular mass of 110 kDa
Conclusions
In this paper we reported that the bifunctional glyoxylate cycle enzyme in E. gracilis had special catalytic features, i.e., its ICL activity was increased by acetyl-CoA. This property was beneficial to sequential ICL and MS reactions under physiological conditions. We also report that EgGCE had an inverted domain structure compared to C. elegans bifunctional GCP. This is an important finding to understand the ancient formation of bifunctional enzymes.
References (32)
Trans-splicing and polycistronic transcription in Caenorhabditis elegans
Trends Genet.
(1995)A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
The crystal structure and active site location of isocitrate lyase from the fungus Aspergillus nidulans
Struct. Fold Des.
(2000) - et al.
Re-examining the role of the glyoxylate cycle in oilseeds
Trends Plant Sci.
(2001) - et al.
Synthesis of reserved polysaccharide from wax esters accumulated as the result of anaerobic energy generation in Euglena gracilis returned from anaerobic to aerobic conditions
Int. J. Biochem.
(1992) - et al.
Bifunctional glyoxylate cycle protein of Caenorhabditis elegans: a developmentally regulated protein of intestine and muscle
Dev. Biol.
(1995) - et al.
Isolation and characterization of isocitrate lyase of castor endosperm
Arch. Biochem. Biophys.
(1982) - et al.
Operation of glyoxylate cycle in halophilic archaea: presence of malate synthase and isocitrate lyase in Haloferax volcanii
FEBS Lett.
(1998) - et al.
Structure of the Escherichia coli malate synthase G: pyruvate:acetyl-coenzyme A abortive ternary complex at 1.95 Å resolution
Protein Sci.
(2003) - et al.
A global analysis of Caenorhabditis elegans operons
Nature
(2002)