A phosphotransferase that generates phosphatidylinositol 4-phosphate (PtdIns-4-P) from phosphatidylinositol and lipid A in Rhizobium leguminosarum - A membrane-bound enzyme linking lipid A and PtdIns-4-P biosynthesis

Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unu sual phosphate-deficient lipid A found in this organism. The enzyme has bee n solubilized with Triton X-100 and purified 80-fold. As shown by co-purifi cation and thermal inactivation studies, the 4'-phosphatase catalyzes not o nly the hydrolysis of (Kdo)(2)-[4'-P-32]lipid IVA but also the transfer the 4'-phosphate of Kdo(2)-[4'-P-32]lipid IVA to the inositol headgroup of pho sphatidylinositol (PtdIns) to generate PtdIns-4-P. Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizob ium meliloti, or the nodulation-defective mutant 24AR of R. leguminosarum. The specific activity for the phosphotransferase reaction is about 2 times higher than that of the 4'-phosphatase, The phosphotransferase assay condit ions are similar to those used for PtdIns kinases, except that ATP and Mg2 are omitted. The apparent K-m for PtdIns is similar to 500 mu M versus 20- 100 mu M for most PtdIns kinases, but the phosphotransferase specific activ ity in crude cell extracts is higher than that of most PtdIns kinases, The phosphotransferase is absolutely specific for the 4-position of PtdIns and is highly selective for PtdIns as the acceptor. The 4'-phosphatase/phosphot ransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns . A phosphoenzyme intermediate may account for the dual function of this en zyme, since a single P-32-labeled protein species (M-r similar to 68,000) c an be trapped and visualized by SDS gel electrophoresis of enzyme preparati ons incubated with Kdo(2)-[4'-P-32]lipid IVA. Although PtdIns is not detect ed in cultures ofR, leguminosarum/etli (CE3), PtdIns may be synthesized dur ing nodulation or supplied by plant membranes, given that soybean PtdIns is an excellent phosphate acceptor. A bacterial enzyme for generating PtdIns- 4-P and a direct link between lipid A and PtdIns-4-P biosynthesis have not been reported previously.