sn-1,2-Diacylglycerol kinase of Escherichia coli. Purification, reconstitution, and partial amino- and carboxyl-terminal analysis

Abstract

The sn-1,2-diacylglycerol kinase structural gene from Escherichia coli was demonstrated to be the dgkA locus previously sequenced (Lightner, V. A., Bell, R. M., and Modrich, P. (1983) J. Biol. Chem. 258, 10856-10861). The dgkA gene product was shown by maxicell analysis to be an Mr = 14,000 membrane-bound protein. When dgkA was placed on a hybrid plasmid under control of the lambda pL promoter, a 100-fold overproduction of diacylglycerol kinase activity was obtained. Diacylglycerol kinase was solubilized from membranes with 2-propanol/heptane/trifluoroacetic acid and purified to near homogeneity by high performance liquid chromatography. Activity was reconstituted in a mixed micellar assay containing beta-octylglucoside, cardiolipin, and sn-1,2-dioleoylglycerol. Amino acid analysis, partial NH2-terminal analysis and COOH-terminal analysis permitted alignment of the polypeptide on the sequenced gene. The data establish that dgkA is the structural gene for the diacylglycerol kinase and establish the primary structure of the enzyme of 122 residues, 13,245 daltons. Secondary structure analysis predicted a protein conformation consisting of three transmembrane alpha-helical segments, an amphipathic helix, and an alpha-helix. Taken together, the predicted helical segments comprise more than 75% of the polypeptide.