| Resistance to classical antibacterial agents attained from the rise of resistant bacterial strains is a major health problem facing the world today. The outer membrane of Gram-negative bacteria is an attractive target for rational attempts to design antimicrobial agents effective solely against Gram-negative bacteria, to which this outer membrane is unique. The molecule 3-deoxy- D-manno-octulosonate (Kdo) is a unique eight-carbon sugar present in the lipopolysaccharide (LPS) layer in the outer membrane of Gram-negative bacteria. The interruption of the synthesis or utilization of Kdo should lead to arrest in cell growth.;The ultimate goal of this dissertation was to gain mechanistic insight into the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate phosphatase (KDP). KDP, the third enzyme in the Kdo pathway, catalyzes the hydrolysis of 3-deoxy-D-manno-octulosonate-8-phosphate to Kdo and inorganic phosphate. A multi-faced approach was employed to fully study and characterize KDP and determine its role in Kdo and LPS biosynthesis. Various techniques in enzymology, molecular biology, biochemistry, and microbiology were incorporated. In chapter II, the characterization of recombinant KDP from a hyperthermophile, Aquifex aeolicus, was undertaken. This was used to validate the similarities of the characteristics of KDP that should exist among different organisms. To gain mechanistic insight into KDP, various techniques were employed. In chapter III, active site mutation studies were conducted to confirm the importance of these residues for catalysis. In chapter IV, the analysis of two thiol phosphate Kdo8P analogues revealed that both these analogues could serve as alternate substrates for KDP. In chapter V, the crystal structure of KDP from E. coli, solved to 2.07 A using molecular replacement, was presented. Finally, in chapter VI, the creation of a DeltaKDP construct in Escherichia coli K-12 provided insight into the in vivo effects of this chromosomal disruption as well as the intracellular location of KDP in E. coli . The kdsC gene does not appear to be an essential gene for the production of Kdo and the viability of E. coli. However, KDP does appear to be inner membrane associated, as predicted by the OPM database and shown via experimental studies. |
