title: Study of arachidonoyl specificity in two enzymes of the PI cycle.
abstract: We identified a conserved pattern of residues L-X((3-4))-R-X((2))-L-X((4))-G, in which -X((n))- is n residues of any amino acid, in two enzymes acting on the polyunsaturated fatty acids, diacylglycerol kinase epsilon (DGKɛ) and phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K Iα). DGKɛ is the only one of the 10 mammalian isoforms of DGK that exhibits arachidonoyl specificity and is the only isoform with the motif mentioned above. Mutations of the essential residues in this motif result in the loss of arachidonoyl specificity. Furthermore, DGKα can be converted to an enzyme having this motif by substituting only one residue. When DGKα was mutated so that it gained the motif, the enzyme also gained some specificity for arachidonoyl-containing diacylglycerol. This motif is present also in an isoform of phosphatidylinositol-4-phosphate-5-kinase that we demonstrated had arachidonoyl specificity for its substrate. Single residue mutations within the identified motif of this isoform result in the loss of activity against an arachidonoyl substrate. The importance of acyl chain specificity for the phosphatidic acid activation of phosphatidylinositol-4-phosphate-5-kinase is also shown. We demonstrate that the acyl chain dependence of this phosphatidic acid activation is dependent on the substrate. This is the first demonstration of a motif that endows specificity for an acyl chain in enzymes DGKε and PIP5K Iα.
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, 1200 Main Street West, Hamilton, Ontario L8N 3Z5, Canada.
authors.forename: Yulia V
authors.initials: YV
authors.lastname: Shulga
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Topham
authors.forename: Richard M
authors.initials: RM
authors.lastname: Epand
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001091:Arachidonate Lipoxygenases / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism*; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000235:genetics / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010712:Phosphatidic Acids / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: C011439:1,2-diacylglycerol; D004075:Diglycerides; D010712:Phosphatidic Acids; D010716:Phosphatidylinositols; D016718:Arachidonic Acid; D001091:Arachidonate Lipoxygenases; D017853:Phosphotransferases (Alcohol Group Acceptor); C526464:DGKE protein, human; D019852:Diacylglycerol Kinase; C020077:1-phosphatidylinositol-4-phosphate 5-kinase