A Deacylase in Rhizobium leguminosarum Membranes That Cleaves the 3-O-Linked β-Hydroxymyristoyl Moiety of Lipid A Precursors

Abstract Lipid A from the nitrogen-fixing bacteriumRhizobium leguminosarum displays many structural differences compared with lipid A of Escherichia coli. R. leguminosarum lipid A lacks the usual 1- and 4′-phosphate groups but is derivatized with a galacturonic acid substituent at position 4′.R. leguminosarum lipid A often contains an aminogluconic acid moiety in place of the proximal glucosamine 1-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E. coli versus R. leguminosarumlipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of laurate and myristate in R. leguminosarum. Recently, we have found that lipid A isolated by pH 4.5 hydrolysis ofR. leguminosarum cells is more heterogeneous than previously reported (Que, N. L. S., Basu, S. S., White, K. A., and Raetz, C. R. H. (1998) FASEB J.12, A1284 (abstr.)). Lipid A species lacking the 3-O-linked β-hydroxymyristoyl residue on the proximal unit contribute to this heterogeneity. We now describe a membrane-bound deacylase from R. leguminosarum that removes a single ester-linked β-hydroxymyristoyl moiety from some lipid A precursors, including lipid X, lipid IVA, and (3-deoxy-d-manno-octulosonic acid)2-lipid IVA. The enzyme does not cleaveE. coli lipid A or lipid A precursors containing an acyloxyacyl moiety on the distal glucosamine unit. The enzyme is not present in extracts of E. coli or Rhizobium meliloti, but it is readily demonstrable in membranes ofPseudomonas aeruginosa, which also contains a significant proportion of 3-O-deacylated lipid A species. Optimal reaction rates are seen between pH 5.5 and 6.5. The enzyme requires a nonionic detergent and divalent metal ions for activity. It cleaves the monosaccharide lipid X at about 5% the rate of lipid IVAand (3-deoxy-d-manno-octulosonic acid)2-lipid IVA. 1H NMR spectroscopy of the deacylase reaction product, generated with lipid IVA as the substrate, confirms unequivocally that the enzyme cleaves only the ester-linked β-hydroxymyristoyl residue at the 3-position of the glucosamine disaccharide.