Membrane phospholipid synthesis in Escherichia coli. Purification, reconstitution, and characterization of sn-glycerol-3-phosphate acyltransferase.

The membrane-bound sn-glycerol-3-phosphate acyltransferase of Escherichia coli was purified to near homogeneity from Triton X-100 extracts of membranes from strain vL3/pvL1 (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) d Biol. Chem. 255, 9413-9420). This strain contains a hybrid plasmid bearing the pZsB gene, a structural gene of the enzyme, and overproduces sn-glycerol-3-phosphate acyltransferase activity more than 10-fold. At each stage of the purification performed in buffers containing Triton X-100, reconstitution of enzyme activity with phospholipids was necessary. Chromatography on Matrex Gel Red A, OctylSepharose CL-4B, and hydroxylapatite yielded preparations containing a single band of 83,000 apparent molecular weight upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Final reconstitutable specific activities in excess of 6 pmol/ min/mg were achieved. Palmitoyl-CoA, oleoyl-CoA, palmitoyl-acyl carrier protein (ACP), and cis-vaccenoyl-ACP thioesters were substrates. The apparent K,,, for sn-glycerol 3-phosphate was 150 p~ with either palmitoyl-CoA or palmitoyl-ACP. Higher V,, values and lower apparent K,,, values were observed for thioesters containing saturated fatty acids than were observed for thioesters containing unsaturated fatty acids. Lysophosphatidic acid was the only product produced. Assay conditions for the reconstituted enzyme were optimized. Bovine serum albumin and additional phospholipids appeared to lessen inhibition by palmitoyl-CoA. Reconstitution with E. coli phospholipids was complete in 10 min at 0°C. Activity depended on the amount and type of phospholipids employed. Some preference for phosphatidylglycerol and cardiolipin was observed. The sn-glycerol-3-phosphate acyltransferase, an integral cytoplasmic membrane protein, contained about 56% hydrophobic amino acid residues.