CLONING OF NOVEL ENZYMES THAT MODIFY LIPID A

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The outer leaflet of the outer membranes of Gram-negative bacteria is almost entirely composed of lipopolysaccharide (LPS) and acts as an efficient permeability barrier against antibacterial agents. Lipid A (endotoxin) functions as the hydrophobic anchor of LPS and is the bioactive component of the molecule leading to Gram-negative septic shock and death. The enzymatic machinery required for lipid A biosynthesis is well understood, however, the enzymes responsible for certain regulated modifications of lipid A remain unknown. These modifications include the addition of 4-aminoarabinose, phosphoethanoiamine, palmitate, 2- hydroxymyristate, phosphate, and the removal of specific fatty acyl chains. It is now known that some of these modifications are important for survival of the bacterium from the host innate immune system. Therefore, understanding the enzymology of these modifications will not only facilitate the understanding of lipid A biosynthesis but will lay the foundation of new molecular insights into pathogenesis. The current study focuses on (I) the characterization of enzymes involved in the addition of 4-aminoarabinose and deacylation of lipid A; (II) structural analysis of the enzymatic products and analysis of their endotoxic activities; and (III) the cloning of these novel genes.