Oxygen requirement for the biosynthesis of the S-2-hydroxymyristate moietyin Salmonella typhimurium lipid A - Function of LpxO, a new Fe2+/alpha-ketoglutarate-dependent dioxygenase homologue

Lipid A molecules of certain Gram-negative bacteria, including Salmonella t yphimurium and Pseudomonas aeruginosa, may contain secondary S-2-hydroxyacy l chains. S. typhimurium has recently been shown to synthesize its S-2-hydr oxymyristate-modified lipid A in a PhoP/PhoQ-dependent manner, suggesting a possible role for the 2-OH group in pathogenesis, We postulated that 2-hyd roxylation might be catalyzed by a novel dioxygenase. Lipid A was extracted from a PhoP-constitutive mutant of S. typhimurium grown in the presence or absence of O-2. Under anaerobic conditions, no 2-hydroxymyristate-containi ng lipid A was formed. Matrix-assisted laser desorption/ionization time-of- flight mass spectrometry of lipid A from cells grown in the presence of O-1 8(2) confirmed the direct incorporation of molecular oxygen into 2-hydroxya cyl-modified lipid A. Using several well characterized dioxygenase protein sequences as probes, tBLASTn searches revealed unassigned open reading fram e(s) with similarity to mammalian aspartyl/asparaginyl beta -hydroxylases i n bacteria known to make 2-hydroxyacylated lipid A molecules. The S. typhim urium aspartyl/asparaginyl beta -hydroxylase homologue (designated lpxO) wa s cloned into pBluescriptSK and expressed in Escherichia coli K-12, which d oes not contain lpxO. Analysis of the resulting construct revealed that lpx O expression is sufficient to induce O-2-dependent formation of 2-hydroxymy ristate-modified lipid A in E. coli. LpxO very likely is a novel Fe2+/alpha -ketoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l ipid A (or of a key precursor). The S. typhimurium lpxO gene encodes a poly peptide of 302 amino acids with predicted membrane-anchoring sequences at b oth ends. We hypothesize that 2-hydroxymyristate chains released from lipop olysaccharide inside infected macrophages might be converted to 2-hydroxymy ristoyl coenzyme A, a well characterized, potent inhibitor of protein N-myr istoyl transferase.