Abstract
Lipid A (also known as endotoxin) is the hydrophobic portion of lipopolysaccharides. It is an essential membrane component required for the viability of gram-negative bacteria. The enzymes involved in its biosynthesis are attractive targets for the development of novel antibiotics. LpxH catalyzes the fourth step of the lipid A biosynthesis pathway and cleaves the pyrophosphate bond of UDP-2,3-diacylglucosamine to yield 2,3-diacylglucosamine 1-phosphate (lipid X) and UMP. Here we present the structures of LpxH from Pseudomonas aeruginosa (PaLpxH). PaLpxH consists of two domains: a catalytic domain that is homologous to the metallophosphoesterases and a helical insertion domain. Lipid X was captured in the crevice between these two domains, with its phosphate group facing the dinuclear metal (Mn2+) center and two acyl chains buried in the hydrophobic cavity. The structures reveal that a large conformational change occurs at the lipid X binding site surface upon the binding/release of the product molecule. Based on these observations, we propose a novel model for lipid X embedding, which involves the scissor-like movement of helix α6, resulting in the release of lipid X into the lipid bilayer.
Highlights
Lipopolysaccharide (LPS) is the main component of the outer membrane of gram-negative bacteria
The lipid X molecule was likely captured in E. coli cells because no substrate molecule (UDP-2,3-diacylglucosamine) or product molecule was added during purification or crystallization
Lipid X is positioned in a crevice between the catalytic and HI domains, with the phosphate group facing the dinuclear metal center and two acyl chains buried in the hydrophobic cavity (Fig. 2)
Summary
Lipopolysaccharide (LPS) is the main component of the outer membrane of gram-negative bacteria. In the fourth step in this pathway, UDP-2,3-diacylglucosamine is hydrolyzed to 2,3-diacylglucosamine-1-phosphate ( called lipid X) and UMP This step is catalyzed by the specific pyrophosphatase LpxH in 70% of gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, and by LpxI in the remaining 30%3–5. These two enzymes provide the same product from the same substrate, they have no sequence similarity and are not found in the same organisms. They have different metal ion requirements and attack different phosphorus atoms during hydrolysis; LpxH attacks the α-phosphate of UDP-2,3-diacylglucosamine, and LpxI attacks the β-phosphate[4]. The relationship of this constituent member with LpxH is discussed
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