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.
Highlights
In many enteric Gram-negative bacteria, including Escherichia coli, lipid A is a hexa-acylated disaccharide of glucosamine, derivatized with phosphate substituents at positions 1 and 4Ј [1, 14, 15]
We have previously demonstrated that R. etli, R. leguminosarum, and Francisella novicida possess selective lipid A 4Ј- and 1-phosphatases (29 –33) that are not present in E. coli and account for the absence of one or both lipid A phosphate groups in these organisms
Given the importance of the phosphate groups of lipid A for Membranes—To prepare extracts for assays, A. tumefaciens its bioactivity and the need for structural studies of the lipid strain C58 was grown at 28 °C in modified LB containing 10 phosphate phosphatases, we report the purification to g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl, and 10 near-homogeneity of a His6-tagged derivative of R. leguminosa- g/ml gentamycin to an A600 of 1.0
Summary
Chemicals and Materials—The [␥-32P] ATP, [glycerol-U-14C]PA, and [U-14C]glycerol-3-phosphate were purchased from PerkinElmer. NaF, NH4VO3, disodium EDTA, diethyl pyrocarbonate (DEPC), HEPES, and MES were obtained from Sigma. Bicinchoninic protein assay reagents [40]. The Non-InterferingTM protein assay was from G-Biosciences. Yeast extract and bactotryptone were from BD Biosciences. Silica Gel 60 TLC plates were obtained from EMD Chemicals
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