An extended β7α7 substrate-binding loop is essential for efficient catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

The enzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the reaction between phosphoenolpyruvate and arabinose 5-phosphate (A5P) in the first committed step in the biosynthetic pathway for the formation of 3-deoxy-D-manno-octulosonate, an important component in the cell wall of Gram-negative bacteria. KDO8P synthase is evolutionarily related to the first enzyme of the shikimate pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase, which uses erythrose 4-phosphate in place of A5P. The A5P binding site in KDO8P synthase is formed by three long loops that extend from the core catalytic (β/α)(8) barrel, β2α2, β7α7, and β8α8. The extended β7α7 loop is always present in KDO8P synthase yet is not observed for DAH7P synthase. Modeling of this loop indicated interactions between this loop and the extended β2α2 loop; both loops provide key hydrogen-bonding contacts with A5P. The two absolutely conserved residues on the β7α7 loop (Gln and Ser) were mutated to Ala in both the metal-dependent KDO8P synthase from Acidithiobacillus ferrooxidans and the metal-independent KDO8P synthase from Neisseria meningitidis. In addition, mutants were constructed for both enzymes with the extended β7α7 loop excised to match the DAH7P synthase architecture. Removal of the loop extension severely hindered efficient catalysis, dramatically increasing the K(m)(A5P) and reducing the k(cat) for both enzymes. Excision of the complete loop was far more detrimental to catalysis than the double mutations of the two conserved Gln and Ser residues. Therefore, the presence of the entire extended β7α7 loop is important for efficient catalysis by KDO8P synthase, with the loop acting to promote efficient and productive binding of A5P.