Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase

Gil Soo Han, Laura O'Hara, Symeon Siniossoglou, George M. Carman

Research output: Contribution to journalArticle

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Abstract

The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2+ or Mg2+ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 °C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent Km = 6.5 mol %) and saturation kinetics with respect to CTP (apparent Km = 0.3mM). dCTP was both a substrate (apparent Km = 0.4 mM) and competitive inhibitor (apparent Ki = 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.

Original languageEnglish (US)
Pages (from-to)20443-20453
Number of pages11
JournalJournal of Biological Chemistry
Volume283
Issue number29
DOIs
StatePublished - Jul 18 2008

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Diacylglycerol Kinase
Cytidine Triphosphate
Yeast
Yeasts
Enzymes
Transferases
dolichol kinase
Diglycerides
Cytidine Diphosphate Diglycerides
Diacylglycerol Cholinephosphotransferase
Kinetics
Ethylmaleimide
Point Mutation
Saccharomyces cerevisiae
Phospholipids
Bacteria
Animals
Genes
Adenosine Triphosphate
Alleles

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Cite this

Han, Gil Soo ; O'Hara, Laura ; Siniossoglou, Symeon ; Carman, George M. / Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase. In: Journal of Biological Chemistry. 2008 ; Vol. 283, No. 29. pp. 20443-20453.
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abstract = "The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2+ or Mg2+ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 °C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent Km = 6.5 mol {\%}) and saturation kinetics with respect to CTP (apparent Km = 0.3mM). dCTP was both a substrate (apparent Km = 0.4 mM) and competitive inhibitor (apparent Ki = 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.",
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Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase. / Han, Gil Soo; O'Hara, Laura; Siniossoglou, Symeon; Carman, George M.

In: Journal of Biological Chemistry, Vol. 283, No. 29, 18.07.2008, p. 20443-20453.

Research output: Contribution to journalArticle

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T1 - Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase

AU - Han, Gil Soo

AU - O'Hara, Laura

AU - Siniossoglou, Symeon

AU - Carman, George M.

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N2 - The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2+ or Mg2+ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 °C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent Km = 6.5 mol %) and saturation kinetics with respect to CTP (apparent Km = 0.3mM). dCTP was both a substrate (apparent Km = 0.4 mM) and competitive inhibitor (apparent Ki = 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.

AB - The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca2+ or Mg2+ ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 °C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent Km = 6.5 mol %) and saturation kinetics with respect to CTP (apparent Km = 0.3mM). dCTP was both a substrate (apparent Km = 0.4 mM) and competitive inhibitor (apparent Ki = 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.

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