Substrate specificity of the pyrophosphohydrolase LpxH determines the asymmetry of lipid A.

Lipopolysaccharides are anchored to the outer membrane of Gram-negative bacteria by a hydrophobic moiety known as lipid A, which potently activates the host innate immune response. Lipid A of , the causative agent of whooping cough, displays unusual structural asymmetry with respect to the length of the acyl chains at the 3 and 3' positions, which are 3OH-C10 and 3OH-C14 chains, respectively. Both chains are attached by the acyltransferase LpxA, the first enzyme in the lipid A biosynthesis pathway, which, in , has limited chain length specificity. However, this only partially explains the strict asymmetry of lipid A. In attempts to modulate the endotoxicity of lipid A, here we expressed the gene encoding LpxA from , which specifically attaches 3OH-C12 chains, in This expression was lethal, suggesting that one of the downstream enzymes in the lipid A biosynthesis pathway in cannot handle precursors with a 3OH-C12 chain. We considered that the UDP-diacylglucosamine pyrophosphohydrolase LpxH could be responsible for this defect as well as for the asymmetry of lipid A. Expression of meningococcal LpxH in indeed resulted in new symmetric lipid A species with 3OH-C10 or 3OH-C14 chains at both the 3 and 3' positions, as revealed by MS analysis. Furthermore, co-expression of meningococcal and resulted in viable cells that incorporated 3OH-C12 chains in lipid A. We conclude that the asymmetry of lipid A is determined by the acyl chain length specificity of LpxH.