Substrate and metal complexes of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifer aeolicus at 1.9-angstrom resolution - Implications for the condensation mechanism

3-Deoxy-D-manno-octulosonate-8-phosphate synthase (KDO8PS) from the hyperth ermophilic bacterium Aquifex aeolicus differs from its Escherichia coli cou nterpart in the requirement of a divalent metal for activity (Duewel, H. S. , and Woodard, R. W. (2000) J. Biol. Chem. 275, 22824-22831), Here we repor t the crystal structure of the A. aeolicus enzyme, which was determined by molecular replacement using E. coli KDO8PS as a model. The structures of th e metal-free and Cd2+ forms of the enzyme were determined in the uncomplexe d state and in complex with various combinations of phosphoenolpyruvate (PE P), arabinose 5-phosphate (A5P), and erythrose 4-phosphate (E4P). Like the E. coli enzyme, A. aeolicus KDO8PS is a homotetramer containing four distin ct active sites at the interface between subunits. The active site cavity i s open in the substrate-free enzyme or when either A5P alone or PEP alone b inds, and becomes isolated from the aqueous phase when both PEP and A5P (or E4P) bind together. In the presence of metal, the enzyme is asymmetric and appears to alternate catalysis between the active sites located on one fac e of the tetramer and those located on the other face. In the absence of me tal, the asymmetry is lost. Details of the active site that may be importan t for catalysis are visible at the high resolution achieved in these struct ures. Most notably, the shape of the PEP-binding pocket forces PEP to assum e a distorted geometry at C-2, which might anticipate the conversion from s p(2) to sp(3) hybridization occurring during intermediate formation and whi ch may modulate PEP reactivity toward A5P. Two water molecules are located in van der Waals contact with the si and re sides of C-2(PEP), respectively . Abstraction of a proton from either of these water molecules by a protein group is expected to elicit a nucleophilic attack of the resulting hydroxi de ion on the nearby C-2(PEP), thus triggering the beginning of the catalyt ic cycle.