Crystal structures of KDOP synthase in its binary complexes with the substrate phosphoenolpyruvate and with a mechanism-based inhibitor

The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate syntha se (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpy ruvate (PEP) and with a mechanism-based inhibitor (K-d = 0.4 muM) were dete rmined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 Angstrom resolution, respectively. Both the KDOPS PEP and KDOPS inhibitor complexes crystallize in the cubic space group I23 with cell constants a = b = c = 117.9 and 117.6 Angstrom, respectively, and one subunit per asymmet ric unit. The two structures are nearly identical, and superposition of the ir Ca atoms indicates an rms difference of 0.41 Angstrom. The PEP in the KD OPS PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results fr om KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of ne gative charge on the carboxylate group of PEP would then suggest that the c ondensation step between PEP and D-arabinose-5-phosphate (A5P) should proce ed in a stepwise fashion through the intermediacy of a transient oxocarbeni um ion at C2 of PEP. The molecular structural results are discussed in ligh t of the chemically similar but mechanistically distinct reaction that is c atalyzed by the enzyme 3-deoxy-D-arabino-2-heptulosonate-7-phosphate syntha se and in light of the preferred enzyme-bound states of the substrate A5P.