TheSaccharomyces cerevisiae ICL2Gene Encodes a Mitochondrial 2-Methylisocitrate Lyase Involved in Propionyl-Coenzyme A Metabolism

Abstract

The Saccharomyces cerevisiae ICL1 gene encodes isocitrate lyase, an essential enzyme for growth on ethanol and acetate. Previous studies have demonstrated that the highly homologous ICL2 gene (YPR006c) is transcribed during the growth of wild-type cells on ethanol. However, even when multiple copies are introduced, ICL2 cannot complement the growth defect of icl1 null mutants. It has therefore been suggested that ICL2 encodes a nonsense mRNA or nonfunctional protein. In the methylcitrate cycle of propionyl-coenzyme A metabolism, 2-methylisocitrate is converted to succinate and pyruvate, a reaction similar to that catalyzed by isocitrate lyase. To investigate whether ICL2 encodes a specific 2-methylisocitrate lyase, isocitrate lyase and 2-methylisocitrate lyase activities were assayed in cell extracts of wild-type S. cerevisiae and of isogenic icl1, icl2, and icl1 icl2 null mutants. Isocitrate lyase activity was absent in icl1 and icl1 icl2 null mutants, whereas in contrast, 2-methylisocitrate lyase activity was detected in the wild type and single icl mutants but not in the icl1 icl2 mutant. This demonstrated that ICL2 encodes a specific 2-methylisocitrate lyase and that the ICL1-encoded isocitrate lyase exhibits a low but significant activity with 2-methylisocitrate. Subcellular fractionation studies and experiments with an ICL2-green fluorescent protein fusion demonstrated that the ICL2-encoded 2-methylisocitrate lyase is located in the mitochondrial matrix. Similar to that of ICL1, transcription of ICL2 is subject to glucose catabolite repression. In glucose-limited cultures, growth with threonine as a nitrogen source resulted in a ca. threefold induction of ICL2 mRNA levels and of 2-methylisocitrate lyase activity in cell extracts relative to cultures grown with ammonia as the nitrogen source. This is consistent with an involvement of the 2-methylcitrate cycle in threonine catabolism.The complete sequencing of the Saccharomyces cerevisiae genome has yielded a large number of open reading frames with unknown function (11). Some of the newly discovered open reading frames exhibited a strong homology with known yeast genes and were demonstrated to encode hitherto-unknown isoenzymes. An example is the PYK2 gene, which encodes a pyruvate kinase isoenzyme but can restore growth of pyk1 null mutants on glucose only when overexpressed (3). In other cases, the biochemical function of the proteins (if any) encoded by the homologous open reading frames remains unknown.An intriguing case is presented by the ICL2 gene. This gene (YPR006c) exhibits a substantial sequence similarity with ICL1 (13), the unique S. cerevisiae structural gene encoding isocitrate lyase (38% identity at the amino acid level). Isocitrate lyase is a key enzyme of the glyoxylate cycle. As this pathway is essential for growth on acetate and ethanol, icl1 null mutants are unable to grow on ethanol or acetate (9, 28). ICL2 is transcribed in ethanol-grown cultures of wild-type S. cerevisiae, and experiments with ICL2-lacZ fusions indicated that its tra...

2‐Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans

“…The enzyme is encoded by the ICL2 gene which is induced using threonine as a nitrogen source. Obviously, this amino acid is degraded via 2-oxobutyrate to propionylCoA followed by oxidation to pyruvate via the methylcitrate cycle [14]. Chemical and enzymatic syntheses of 2-methylisocitrate revealed that the substrate for both 2-methylisocitrate lyases is the same enantiomer of the threo-diastereomeric pair.…”

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

“…The S. cerevisiae genome does not encode a propionyl-CoA carboxylase and therefore cannot catabolize propionyl-CoA via the methylmalonyl-CoA pathway. However, there is evidence that propionylCoA is catabolized via the methylcitrate cycle in S. cerevisiae (69,70). No gene has been associated with the reported bacterial 2HG synthase activity yet, hampering the search for a 2HG synthase candidate gene in other genomes.…”

Global Transcription Analysis of Krebs Tricarboxylic Acid Cycle Mutants Reveals an Alternating Pattern of Gene Expression and Effects on Hypoxic and Oxidative Genes

“…The methylcitrate cycle is used for the assimilation of propionate via propionyl-CoA, which is generated from the oxidation of odd-chain fatty acids or amino acids, such as threonine or methionine (Luttik et al, 2000). The methylcitrate cycle genes are induced by propionate (Epstein et al, 2001a) and are not expressed under anaerobic conditions in a glucose-limited chemostat culture (Ter Linde et al, 1999).…”

“…The function and species distribution of the methylcitrate cycle are still poorly understood, but this pathway is intimately linked to the TCA cycle. The three distinct enzymes of this mitochondrial pathway: methylcitrate synthase, methylaconitate dehydratase, and methylisocitrate lyase, appear to be encoded by the CIT3, PDH1, and ICL2 genes, respectively (Luttik et al, 2000;Horswill and Escalante-Semerena, 2001). Expression of PDH1 was altered in 13 of the 15 mutant strains, and CIT3 and ICL2 were also highly responsive to TCA cycle defects.…”