Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

Abstract

The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca 2؉ -dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded ␤-barrel, reveals that the active site is located between the barrel wall and an ␣-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca 2؉ forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.crystal structure ͉ endotoxin ͉ lipopolysaccharide ͉ outer-membrane protein ͉ phospholipase

Modulating the innate immune response by combinatorial engineering of endotoxin

“…For example, the crystal structure of LpxR and modeling of the structure with lipid A did not indicate any important interactions of the enzyme with the 1-phosphate group of lipid A (25). The enzyme is Ca 2+ -dependent, and the 4'-phosphate group interacts with this cation.…”

Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity

“…Most OM phospholipases share an integral membrane beta-barrel structure with an active site exposed to the outer leaflet [73,75,76]. In response to OM damage [77,78], or PhoPQ activation [29,78], the conserved PagP enzyme transfers palmitoyl groups from the sn-1 position of GPL to lipid A using an outer leaflet active site (Fig.…”

Structural basis for catalysis at the membrane-water interface

“…The resulting tetra-acylated lipid A has lower immunological activity (endotoxicity), thus facilitating evasion from the innate immune response of the host. Interestingly, in H. pylori LpxR is constantly activated leading to conversion of the entire lipid A population into the tetra-acylated form [113, 114]. …”

“…LpxR from Salmonella enterica ( Se LpxR) was solved by x-ray crystallography to 1.9 Å resolution [114]. The structure revealed a 12-stranded antiparallel β-barrel with an unusually long periplasmic turn 4 that forms a loop inside the β-barrel effectively sealing off the fold from the periplasmic side.…”