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J. Biol. Chem., Vol. 283, Issue 29, 20443-20453, July 18, 2008

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Characterization of the Yeast DGK1-encoded CTP-dependent Diacylglycerol Kinase^*

Gil-Soo Han, Laura O'Hara, Symeon Siniossoglou, and George M. Carman¹

From the Department of Food Science and the Rutgers Center for Lipid Research, Rutgers University, New Brunswick, New Jersey 08901 and the Cambridge Institute for Medical Research, University of Cambridge, Hills Road, CB2 0XY Cambridge, United Kingdom

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 K_m = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K_m = 0.3 mM). dCTP was both a substrate (apparent K_m = 0.4 mM) and competitive inhibitor (apparent K_i = 0.4 mM) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.

Received for publication, April 15, 2008

^* This work was supported, in whole or in part, by National Institutes of Health Grant GM-28140 (to G. M. C.) from the United States Public Health Service. This work was also supported by a Wellcome Trust Career Development Fellowship in Basic Biomedical Science (to S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This article was selected as a Paper of the Week.

¹ To whom correspondence should be addressed: Dept. of Food Science, Rutgers University, 65 Dudley Rd., New Brunswick, NJ 08901. Tel.: 732-932-9611 (ext. 217); E-mail: carman{at}aesop.rutgers.edu.

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