The lipopolysaccharide (LPS), also known as endotoxin, is a unique constituent of the outer cell membrane of Gram negative bacteria and is responsible for the pathophysiological phenomena of the shock syndrome associated with Gram negative sepsis. Since LPS is essential for bacterial growth, the enzymes that catalyze its synthesis are important drug targets for antimicrobial chemotherapy. One of the principal components of LPS is 3-deoxy-D-manno-octulosonate (KD0). This 8-carbon sugar is first synthesized as a phosphorylated precursor (KDO8P) by a specific bacterial synthase. The reaction, with involves the condensation of phosphoenolpyruvate (PEP) with arabinose 5-(A5P) to yield KDO8P and Pi, is poorly understood at present. This fact has limited the suitability of KDO8P synthase as therapeutic target. The current project will employ structural analysis of KDO8P synthase from Escherichia coli to determine the details of the enzyme catalytic mechanism. The three-dimensional structure of the wild type enzyme will be solved first in the absence of bound ligands. Subsequent structure determinations will be done in the presence of the enzyme substrates (PEP+ a5P), its products (KDO8P, Pi) and analogs of these ligands, and will provide initial information on the amino acids in the active site of KDO8P synthase that are important for catalysis. The catalytic role of chemical groups of KDO8P synthase will be examined further in structures obtained at different pH values, and by employing mutant forms of the enzyme. Notably, E. coli KDO8P synthase exhibits sequence similarity to two other enzymes that recognize a pyruvyl moiety, the bifunctional 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase/chorismate mutase from Bacillus subtilis and the chorismate mutase from Staphylococcus xylosus, for which no structural information is available. Thus, studies of KDO8P synthase will provide a mechanism model for a new class of enzymes that catalyze the transfer of three- carbon units.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29AI042868-02
Application #
2887709
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Heyse, Stephen P
Project Start
1998-05-01
Project End
2003-04-30
Budget Start
1999-05-01
Budget End
2000-04-30
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Wayne State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202