The lipopolysaccharide (LPS) of Gram negative bacteria, also known as endotoxin, is responsible for the pathophysiological phenomena of the shock syndrome associated with Gram negative sepsis. 3-Deoxy-Dmanno-octulosonate (KDO) is an essential constituent of the LPS of all Gram negative bacteria. This eight carbon sugar is the first component of the oligosaccharide core region that links the lipid A moiety of LPS to the O-antigen. The combination of lipid A and two KDO units is the LPS component where the endotoxin activity is located. The first step in the synthesis of KDO is the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO 8-phosphate (KDO8P), the phosphorylated precursor of KDO, catalyzed by KDO8P synthase (KDO8PS). Since LPS is essential for bacterial growth, this enzyme is a potential target for new antimicrobial drugs. There are two classes of highly homologous KDO8PS's differing primarily in the requirement, or lack thereof, of a metal ion for activity. We have determined the structure of one member of the metal-free class, the Escherichia coil enzyme, and of one member of the metal-requiring class, the enzyme from the hyperthermophile Aquifex aeolicus. These studies have revealed that KDO8PS is a homotetramer with two active sites located on each face of the enzyme. There is alternation of catalysis between the two faces of the enzyme, such that when PEP and A5P bind and react in the active sites located on one face, only PEP binds at the active sites located on the other face. Despite the wealth of information derived from the initial structural and biochemical studies, the mechanism of the reaction catalyzed by KDO8PS is not clear yet. The current project will employ methods of kinetic analysis, spectroscopy, rational mutagenesis, directed evolution and X-ray diffraction to investigate (a) the function of individual residues and active site water in catalysis, and the mechanistic differences between metallo and non-metallo synthases, (b) the molecular mechanism of alternating face catalysis.