Structure and inhibition of enzymes that assemble outer membrane lipids

Abstract

The outer surface of Gram-negative bacteria is covered with a remarkable, macro-molecular glycolipid known as lipopolysaccharide (LPS), the hydrophobic anchor of which is lipid A. In Escherichia coli, lipid A consists of a hexa-acylated disaccharide of glucosamine with phosphate groups at the 1 and 4′ positions. Inhibition of any of the first seven enzymes that synthesize lipid A is lethal. These enzymes are expressed constitutively and are conserved in virtually all Gram-negative bacteria. The structures of the first three have been determined. Potent inhibitors have recently been discovered for the second enzyme (LpxC), with antibiotic activity comparable to ciprofloxacin. These compounds are slow, tight-binding inhibitors that interact with the active site zinc ion and fatty acid binding tunnel of LpxC. Nine constitutive and ten inducible enzymes in E. coli are needed for lipid A biosynthesis and modification, respectively. The ABC transporter MsbA, related to eucaryotic Mdr proteins, flips newly made LPS across the inner membrane. MsbA is also required for LPS and phospholipid export to the outer membrane. Since lipid A modification enzymes are extra-cellular, they are convenient reporters for following LPS movement through different layers of the cell envelope. Lipid A (endotoxin) is the active component of LPS that stimulates immune cells. The signaling receptor for lipid A is the TLR4 protein, which is distantly related to the IL-1 receptor. TLR4 and its subunit MD2 are activated by some types of lipid A but antagonized by others. One can re-engineer the structure of lipid A in viable bacteria to be a TLR4 antagonist (or partial agonist), with applications in vaccine development. (Supported by NIH grants GM-51310 and GM-51796 to C.R.H.R.)