Identification of a broad family of lipid A late acyltransferases with non‐canonical substrate specificity

Summary Most Gram‐negative organisms produce lipopolysaccharide ( LPS ), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the R aetz pathway, a conserved nine‐step enzymatic process first characterized in E scherichia coli . The E psilonproteobacterium H elicobacter pylori uses the R aetz pathway to synthesize lipid A ; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp 0255, which transfers a secondary acyl chain to the 3′‐linked primary acyl chain of lipid A , an activity similar to that of E . coli LpxM . This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM , catalyses addition of a C 12:0 or C 14:0 acyl chain to the 3′‐linked primary acyl chain of lipid A , complementing an E . coli   LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologues in C ampylobacter jejuni and W olinella succinogenes can act before the 2′ secondary acyltransferase, LpxL , as well as the 3‐deoxy‐ d ‐ manno ‐octulosonic acid ( K do) transferase, KdtA . Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E . coli   LpxM homologue, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homologue.