Elucidation of Lipopolysaccharide Biosynthesis through the Activity of CMPKdo Synthase with 3-deoxy-D-mannooctulosonic acid and its 5-deoxy Derivative
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The outer membrane of gram-negative bacteria contains lipopolysaccharide (LPS), an amphipathic macromolecule which plays a critical role in antibiotic resistance. Bacteria with shortened LPS have been shown to be much more susceptible to some antibiotics. Critical to the biosynthesis of LPS is the eight carbon sugar 3-deoxy-Dmanno- octulosonic acid (Kdo), which forms the bridge between the hydrophobic membrane bound lipid A portion and the hydrophilic core and O-antigen regions. Prior to its incorporation into LPS, Kdo must be activated by CMP-Kdo synthetase (KdsB), without which bacteria would have a truncated LPS, leading to a loss of the hydrophilic chain. In this experiment, Kdo and its 5-deoxy derivative have been synthesized via the Cornforth method to gain insight into the importance of three amino acid residues found in the active site of KdsB (Gln 98, Tyr 185, and Glu 210) which could play a key role in binding and orientation of Kdo. 5-deoxy-Kdo showed some, yet much lower activity than Kdo in E. coli KdsB, suggesting that the interaction between these residues and the fifth hydroxyl group of Kdo are critical in the mechanism of Kdo activation. Additionally, Kdo showed no activity in S. oneidensis KdsB in comparison to 8-amino-Kdo, suggesting that this enzyme is responsible for the selectivity of the 8-amino-Kdo sugar present in the core region of S.oneidensis LPS.