Purification and characterization of the lipid A 1‐phosphatase (LpxE) from Francisella novicida

Abstract

The outer monolayer of the gram‐negative outer membrane is composed of the diglucosamine saccharolipid lipid A, the hydrophobic anchor of lipopolysaccharide. The lipid A of E. coli is derivatized with phosphate moieties at the 1‐ and 4′‐positions. Removal of the 1‐phosphate of lipid A results in low‐level polymyxin resistance and reduced activation of the human TLR4/MD‐2 innate immune receptor. Some gram‐negative species, including Francisella novicida, enzymatically remove the 1‐phosphate by LpxE, a lipid A 1‐phosphatase. To understand LpxE of Francisella novicida on a mechanistic level, the enzyme has been purified to homogeneity. A highly sensitive, in vitro phosphatase activity assay was developed. LpxE shows optimum in vitro activity at a pH of 5, consistent with the proposed mechanism of integral membrane lipid phosphate phosphatases. Alanine point mutants of the eleven conserved lipid phosphate phosphatase residues have been constructed. Four point mutants, R142A, S169A, R204A, and D214A are inactive, whereas H210A and H171A are 500‐ and 100,000‐fold less active than wild‐type, respectively. The effect of divalent cations and detergent concentration on activity has also been determined. Francisella LpxE activity is highly dependent upon the presence of two 3‐deoxy‐d‐manno‐oct‐2‐ulsonic acid residues in the 6’‐position of lipid A. This research was supported by NIH grant GM51796 to C.R.H. Raetz.