title: Purification and characterization of the lipid A 1-phosphatase LpxE of Rhizobium leguminosarum.
abstract: LpxE, a membrane-bound phosphatase found in Rhizobium leguminosarum and some other Gram-negative bacteria, selectively dephosphorylates the 1-position of lipid A on the outer surface of the inner membrane. LpxE belongs to the family of lipid phosphate phosphatases that contain a tripartite active site motif and six predicted transmembrane helices. Here we report the purification and characterization of R. leguminosarum LpxE. A modified lpxE gene, encoding a protein with an N-terminal His6 tag, was expressed in Escherichia coli. The protein was solubilized with Triton X-100 and purified to near-homogeneity. Gel electrophoresis reveals a molecular weight consistent with the predicted 31 kDa. LpxE activity is dependent upon Triton X-100, optimal near pH 6.5, and Mg2+-independent. The H197A and R133A substitutions inactivate LpxE, as does treatment with diethyl pyrocarbonate. In a mixed micelle assay system, the apparent Km for the precursor lipid IV(A) is 11 microm. Substrates containing the 3-deoxy-d-manno-oct-2-ulosonic acid disaccharide are dephosphorylated at similar rates to lipid IV(A), whereas glycerophospholipids like phosphatidic acid or phosphatidylglycerol phosphate are very poor substrates. However, an LpxE homologue present in Agrobacterium tumefaciens is selective for phosphatidylglycerol phosphate, demonstrating the importance of determining substrate specificity before assigning the functions of LpxE-related proteins. The availability of purified LpxE will facilitate the preparation of novel 1-dephosphorylated lipid A molecules that are not readily accessible by chemical methods.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Karbarz
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: David A
authors.initials: DA
authors.lastname: Six
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710. Electronic address: raetz@biochem.duke.edu.
authors.forename: Christian R H
authors.initials: CRH
authors.lastname: Raetz
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004789:Enzyme Activation / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008050:Lipid A / Q000737:chemistry / Q000378:metabolism; D020125:Mutation, Missense; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D008050:Lipid A; D011994:Recombinant Proteins; C478996:lipid A 1-phosphatase, Rhizobium leguminosarum; D010744:Phosphoric Monoester Hydrolases