DOMAINS OF ESCHERICHIA-COLI ACYL CARRIER PROTEIN IMPORTANT FOR MEMBRANE-DERIVED-OLIGOSACCHARIDE BIOSYNTHESIS

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthes is, lipopolysaccharide biosynthesis, activation of prohemolysin, and m embrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assembl e an acyl chain or to transfer an acyl group from the thioester linkag e to a specific substrate. By contrast, the phosphopantetheine prosthe tic group is not required for membrane-derived oligosaccharide biosynt hesis, and the function of acyl carrier protein in this biosynthetic s cheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier prote in or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and cons equent loss of activity in the membrane glucosyltransferase reaction o f membrane-derived oligosaccharide biosynthesis. These peptide fragmen ts, however, inhibited the action of intact acyl carrier protein in th e enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need far at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacter ia (including representatives from all four subgroups) and characteriz ed the proteins as active or inhibitory in the membrane glucosyltransf erase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mu tagenesis to change amino acids conserved in active, and altered in in active, acyl carrier proteins suggest the importance of residues Glu 4 , Glu-14, Glu-21, and Asp-51. The first 3 of these residues define a f ace of acyl carrier protein that includes the beginning of the loop re gion, residues 16 to 36. Additionally, screening for membrane glucosyl transferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most clo sely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesi zed by the mechanism found in E. coli.