Covalent Binding of Lipid to Protein: Diglyceride and Amide‐Linked Fatty Acid at the N‐Terminal End of the Murein‐Lipoprotein of the Escherichia coli Outer Membrane

Abstract

A new structure of a lipid and its covalent linkage to a protein (murein‐lipoprotein of the Escherichia coli outer membrane) is described. Glycerylcysteine (S‐(propane‐2′,3′‐diol)‐3‐thio‐2‐aminopropanic acid) at the N‐terminal end of the polypeptide chain is the attachment site of two ester‐bound fatty acids. An additional fatty acid is bound as amide to the N‐terminal group. The diglyceride residue on the cysteine could be derived from the phospholipid pathway since the fatty acid composition is very similar to that of the phospholipids from the same cells. In contrast, 65% of the amide‐linked fatty acid is palmitate. The main fatty acids are palmitic acid (53%), cis‐vaccenic acid (20.7%), 9,10‐methylene‐hexadecanoic acid (10.6%) and palmitoleic acid (9.4%). The structure of glycerylcysteine was established by chemical degradation of the compound containing the fatty acids, by incorporation studies of [35 S]sulfate, [35 S]cysteine, [14 C]cystine, [methyl‐3 H]methionine and [2‐3 H]glycerol and by chemical synthesis. Glycerylcysteine was isolated as a constituent of the peptide (Ser)‐Ser‐Am‐Ala‐Lys. The C‐terminal part of the lipopeptide sequence overlaps with the N‐terminal end of the known sequence of the polypeptide chain. Its N‐terminal position was confirmed by isolation of the N‐terminal polypeptide fragment (position 1–31) after cleavage with cyanogen bromide. This contained the lipid.