Glucose-induced inactivation of isocitrate lyase in is mediated by the cAMP-dependent protein kinase catalytic subunits Tpk 1 and Tpk2

Departamento de Bioquimica y Biologia Molecular e Instituto Universitario de Biotecnologia de Asturias (IUBA), Universidad de Oviedo, 33006 Oviedo, Spain
FEBS Letters (Impact Factor: 3.17). 04/1996; 385(1-2):43-46. DOI: 10.1016/0014-5793(96)00344-4


Glucose-induced inactivation of isocitrate lyase (Icl) has been related to protein phosphorylation. Moreover, since rapid reversible inactivation preceded irreversible inactivation of the enzyme, phosphorylation was proposed as the triggering reaction that makes the enzyme accessible to the proteolytic machinery. The protein kinase involved in the process is unknown at the moment. In this work we demonstrate that Tpk1 and Tpk2, the catalytic subunits of cAMP-dependent protein kinase, are involved in the signalling of short-term and long-term inactivation processes of Icl. We also demonstrate that threonine 53 is involved in a regulatory mechanism necessary for short-term reversible inactivation of Icl, probably mediated through its phosphorylation. Other, as yet unidentified, residues are likely to be the target of distinct protein kinases mediating the irreversible long-term inactivation of Icl.

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    • "However, this activity was much lower than the activity achieved during growth on mixture of ethanol and d-glycerol. This observation - in addition to the role of glucose repression [18,33] - is possibly explained by glucose-induced post-translational modifications such as phosphorylation [34,35] or proteolytic inactivation [36], which have been shown to regulate Icl1p activity. "
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    ABSTRACT: Glycolic acid is a C2 hydroxy acid that is a widely used chemical compound. It can be polymerised to produce biodegradable polymers with excellent gas barrier properties. Currently, glycolic acid is produced in a chemical process using fossil resources and toxic chemicals. Biotechnological production of glycolic acid using renewable resources is a desirable alternative. The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are suitable organisms for glycolic acid production since they are acid tolerant and can grow in the presence of up to 50 g l-1 glycolic acid. We engineered S. cerevisiae and K. lactis for glycolic acid production using the reactions of the glyoxylate cycle to produce glyoxylic acid and then reducing it to glycolic acid. The expression of a high affinity glyoxylate reductase alone already led to glycolic acid production. The production was further improved by deleting genes encoding malate synthase and the cytosolic form of isocitrate dehydrogenase. The engineered S. cerevisiae strain produced up to about 1 g l-1 of glycolic acid in a medium containing d-xylose and ethanol. Similar modifications in K. lactis resulted in a much higher glycolic acid titer. In a bioreactor cultivation with d-xylose and ethanol up to 15 g l-1 of glycolic acid was obtained. This is the first demonstration of engineering yeast to produce glycolic acid. Prior this work glycolic acid production through glyoxylate cycle has only been reported in bacteria. The benefit of yeast host is the possibility for glycolic acid production also in low pH which was demonstrated in flask cultivations. Production of glycolic acid was first shown in S. cerevisiae. To test whether a Crabtree negative yeast would be better suited for glycolic acid production we engineered K. lactis in the same way and demonstrated it to be a better host for glycolic acid production.
    Full-text · Article · Sep 2013 · Microbial Cell Factories
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    • "This is consistent with the observation that phosphorylation of FbPase by PKA is strongly activated by fructose-2,6-bispho- sphate, a regulatory metabolite that is formed when glucose is metabolized (Gancedo et al., 1983). While partial inactivation of isocitrate lyase by glucose is also dependent on phosphorylation by PKA (Ordiz et al., 1996), there is no information on the protein kinase(s) performing the phosphorylation of the malate dehydrogenase isoenzyme Mdh2 "
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    ABSTRACT: In the presence of glucose, yeast undergoes an important remodelling of its metabolism. There are changes in the concentration of intracellular metabolites and in the stability of proteins and mRNAs; modifications occur in the activity of enzymes as well as in the rate of transcription of a large number of genes, some of the genes being induced while others are repressed. Diverse combinations of input signals are required for glucose regulation of gene expression and of other cellular processes. This review focuses on the early elements in glucose signalling and discusses their relevance for the regulation of specific processes. Glucose sensing involves the plasma membrane proteins Snf3, Rgt2 and Gpr1 and the glucose-phosphorylating enzyme Hxk2, as well as other regulatory elements whose functions are still incompletely understood. The similarities and differences in the way in which yeasts and mammalian cells respond to glucose are also examined. It is shown that in Saccharomyces cerevisiae, sensing systems for other nutrients share some of the characteristics of the glucose-sensing pathways.
    Preview · Article · Aug 2008 · FEMS Microbiology Reviews
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    • "A similar site has been found in C. cinereus acu7 (Chaure et al., 1997), and ICL1 sequences of S. cerevisiae (Fernández et al., 1992) and Candida tropicalis (Atomi et al., 1990). In particular, S. cerevisiae ICL1 has been proposed to be phosphorylated at T53, followed by inactivation in the presence of glucose (Ló pez-Boado et al., 1988; Ordiz et al., 1996). However, the role of the motif KRGT in FPICL1 remains to be assessed. "
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    ABSTRACT: This study investigated the subcellular localization of key enzymes of the glyoxylate cycle, i.e. isocitrate lyase (ICL; EC and malate synthase (EC, that function constitutively in coordination with oxalate biosynthesis of glucose-grown Fomitopsis palustris. The ICL purified previously from F. palustris is termed FPICL1. Subcellular fractionation analysis of the cell homogenate by the sucrose density-gradient method showed that both key enzymes were present in peroxisomes, whereas acetyl-CoA synthase (EC and oxalate-producing oxaloacetate acetylhydrolase (EC were cytosolic. The peroxisomal localization of FPICL1 was further confirmed by electron microscopic and immunocytochemical analysis with anti-FPICL1 antibody. In addition, the peroxisomal target signal, composed of SKL at the C terminus of the cDNA encoding FPICL1, was found, which also suggests that FPICL1 is peroxisomal. Accordingly, it is postulated that transportation of succinate from peroxisomes to mitochondria, and vice versa, for the transportation of isocitrate or citrate, occurs in glucose-grown F. palustris for the constitutive metabolic coordination of the TCA and glyoxylate cycles with oxalate biosynthesis.
    Preview · Article · Jul 2006 · Microbiology
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