Structural and mechanistic investigation of 3-deoxy-D-manno-octulosonate-8-phosphate synthase by solid-state REDOR NMR.
15N¿(31)P¿ REDOR NMR experiments were applied to lyophilized binary complexes of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS), with each of its natural substrates, phosphoenolpyruvate (PEP) and arabinose-5-phsophate (A5P), and with a mechanism-based inhibitor (K(i) = 0.4 microM), directly characterizing the active site basic residues involved in the binding of their carboxylate and phosphate moieties. KDO8PS was labeled uniformly with (15)N or [eta-(15)N(2)]Arg, and the ligands were selectively labeled with (13)C and (15)N. The NMR data established that PEP is bound by KDO8PS via a preserved set of structurally rigid and chemically unique Arg and Lys residues, with 5 A (upper limit) between epsilon-(15)N of this Lys and (31)P of PEP. A5P is bound in its cyclic forms to KDO8PS via a different set of Lys and Arg residues. The two sets arise from adjacent subsites that are capable of independent and sufficiently strong binding. The inhibitor is best characterized as an A5P-based substrate analogue inhibitor of KDO8PS. Five mutants in which highly conserved arginines were replaced with alanines were prepared and kinetically characterized. Our solid-state NMR observations complement the crystallographic structure of KDO8PS, and in combination with the mutagenesis results enable tentative assignment of the NMR-identified active site residues. Lys-138 and Arg-168 located at the most recessed part of the active site cavity are the chemically distinct and structurally rigid residues that bind PEP phosphate; R168A resulted in 0.1% of wild-type activity. Arg-63, exposed at the opening of the active site barrel, is the flexible residue with a generic chemical shift that binds A5P; R63A resulted in complete deactivation. The mechanistic implications of our results are discussed.
Kaustov
L
L
Department of Chemistry, Institute of Catalysis Science and Technology, and Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Kababya
S
S
Du
S
S
Baasov
T
T
Gropper
S
S
Shoham
Y
Y
Schmidt
A
A
D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001059:Apoproteins / Q000737:chemistry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010428:Pentosephosphates / Q000737:chemistry*; D010728:Phosphoenolpyruvate / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013400:Sugar Acids; D013403:Sugar Phosphates
C121801:3-deoxy-manno-octulosonate-8-phosphate; D001059:Apoproteins; D010428:Pentosephosphates; D011994:Recombinant Proteins; D013400:Sugar Acids; D013403:Sugar Phosphates; C028055:arabinose 5-phosphate; D010728:Phosphoenolpyruvate; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases