A deacylase in Rhizobium leguminosarum membranes that cleaves the 3-O-linked beta-hydroxymyristoyl moiety of lipid A precursors

Lipid A from the nitrogen-fixing bacterium Rhizobium 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 der ivatized with a galacturonic acid substituent at position 4', R, leguminosa rum lipid A often contains an aminogluconic acid moiety in place of the pro ximal glucosamine l-phosphate unit. Striking differences also exist in the secondary acyl chains attached to E, coli versus R, leguminosarum lipid A, specifically the presence of 27-hydroxyoctacosanoate and the absence of lau rate and myristate in R. leguminosarum, Recently, we have found that lipid A isolated by pH 4.5 hydrolysis of R, leguminosaram cells is more heterogen eous than previously reported (Que, N, L, S,, Basu, S, S., White, K, A., an d Raetz, C. R. H. (1998) FASEB J. 12, A1284 (abstr.)), Lipid A species lack ing the 3-O-linked beta-hydroxymyristoyl residue on the proximal unit contr ibute to this heterogeneity. We now describe a membrane-bound deacylase fro m R, leguminosarum that removes a single ester-linked beta-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 cleave E. 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 of Pseud omonas aeruginosa, which also contains a significant proportion of 3-O-deac ylated 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 a ctivity. It cleaves the monosaccharide lipid X at about 5% the rate of lipi d IVA and (3-deoxy-D-manno-octulosonic acid)(2)-lipid IVA. H-1 MMR spectros copy of the deacylase reaction product, generated with lipid IVA as the sub strate, confirms unequivocally that the enzyme cleaves only the ester-linke d beta-hydroxymyristoyl residue at the 3-position of the glucosamine disacc haride.