|M.Sc Thesis||Department of Chemistry|
|Supervisor:||Prof. Adir Noam|
The enzyme KDO8Ps (3-Deoxy-D-Manno-Octulosonate 8-Phosphate Synthase) catalyzes the condensation reaction between PEP (phosphoenolpyruvate ) and A5P (arabinose 5-phosphate) to form KDO8P and inorganic phosphate.
This important enzymatic reaction controls the carbon flow in the biosynthetic formation of a phoshorylated precursor of the unique monosaccharide KDO, which is an essential constituent of the lipopolysaccharide of all Gram-negative bacteria and plays a crucial role in their assembly process.
The knowledge of the three-dimensional structure with substrates will aid in the design of drugs to treat infection caused by antibiotic resistant Gram-negative bacteria. The aim of this study is to determine the three-dimensional structure of the enzyme KDO8PS from E. coli binary complexes with A5P, E and Z fluorophosphoenolpyruvate (Z-FPEP and E-FPEP ).
The crystal structures of KDO8Ps with different substrates have been previously determined. In the course of this work we obtained crystals of the following mentioned above enzyme complexes: KDO8P:Z-FPEP , KDO8P:E-FPEP and KDO8P:A5P, which crystallized in the cubic space group I23 with cell constants of =119.4 Å, 118.02 Å and 117.44 Å, respectively.
Crystallographic phases were determined using molecule replacement. These protein structures are in the process of refinement. In our work we present two structures of KDO8Ps with Pi and Z-FPEP that were obtained from crystal structure determination of KDO8P + Z-FPEP ( Z-FPEP in active site ) and KDOAPI ( Pi in active site ). This structural information allows us to make a set of statements about the catalytic mechanism of KDO8Ps.