Structural and mechanistic investigation of 3-deoxy-D-manno-octulosonate-8-phosphate synthase by solid-state REDOR NMR

N-15{P-31} REDOR NMR experiments were applied to lyophilized binary complex es of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with ea ch of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phs ophate (A5P), and with a mechanism-based inhibitor (K-i = 0.4 muM), directl y characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with N-15 or [eta-N-15(2)]Arg, and the ligands were selectively labeled with C- 13 and N-15. The NMR data established that PEP is bound by KDO8PS via a pre served set of structurally rigid and chemically unique Arg and Lys residues , with 5 Angstrom (upper limit) between epsilon-N-15 of this Lys and P-31 o f PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Ly s and Arg residues. The two sets arise from adjacent subsites that are capa ble of independent and sufficiently strong binding. The inhibitor is best c haracterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five m utants: in which highly conserved arginines were replaced with alanines wer e prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination wit h the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed par t of the active site cavity are the chemically distinct and structurally ri gid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type a ctivity. Arg-63, exposed at the opening of the active site barrel, is the f lexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.