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J Biol Chem, Vol. 274, Issue 16, 11139-11149, April 16, 1999

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

Shib Sankar Basu, John D. York^§, and Christian R. H. Raetz

From the Departments of  Biochemistry and ^§ Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Membranes of Rhizobium leguminosarum contain a 3-deoxy-D-manno-octulosonic acid (Kdo)-activated lipid A 4'-phosphatase required for generating the unusual phosphate-deficient lipid A found in this organism. The enzyme has been solubilized with Triton X-100 and purified 80-fold. As shown by co-purification and thermal inactivation studies, the 4'-phosphatase catalyzes not only the hydrolysis of (Kdo)₂-[4'-³²P]lipid IV_A but also the transfer the 4'-phosphate of Kdo₂-[4'-³²P]lipid IV_A to the inositol headgroup of phosphatidylinositol (PtdIns) to generate PtdIns-4-P. Like the 4'-phosphatase, the phosphotransferase activity is not present in Escherichia coli, Rhizobium 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 conditions are similar to those used for PtdIns kinases, except that ATP and Mg²⁺ are omitted. The apparent K_m for PtdIns is ~500 µM versus 20-100 µM for most PtdIns kinases, but the phosphotransferase specific activity 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/phosphotransferase can be eluted from heparin- or Cibacron blue-agarose with PtdIns. A phosphoenzyme intermediate may account for the dual function of this enzyme, since a single ³²P-labeled protein species (M_r ~68,000) can be trapped and visualized by SDS gel electrophoresis of enzyme preparations incubated with Kdo₂-[4'-³²P]lipid IV_A. Although PtdIns is not detected in cultures of R. leguminosarum/etli (CE3), PtdIns may be synthesized during 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.

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