title: Structures of Aquifex aeolicus KDO8P synthase in complex with R5P and PEP, and with a bisubstrate inhibitor: role of active site water in catalysis.
abstract: We have determined the crystal structures of the metalloenzyme 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase from Aquifex aeolicus in complex with phosphoenolpyruvate (PEP) and ribose 5-phosphate (R5P), and with a bisubstrate inhibitor that mimics the postulated linear reaction intermediate. R5P, which is not a substrate for KDO8P synthase, binds in a manner similar to that of arabinose 5-phosphate (A5P), which is the natural substrate. The lack of reactivity of R5P appears to be primarily a consequence of the loss of a water molecule coordinated to Cd(2+) and located on the si side of PEP. This water molecule is no longer present because it cannot form a hydrogen bond with C2-OH(R5P), which is oriented in a different direction from C2-OH(A5P). The bisubstrate inhibitor binds with its phosphate and phosphonate moieties occupying the positions of the phosphate groups of A5P and PEP, respectively. One of the inhibitor hydroxyls replaces water as a ligand of Cd(2+). The current work supports a mechanism for the synthesis of KDO8P, in which a hydroxide ion on the si side of PEP attacks C2(PEP), forming a tetrahedral-like intermediate with a buildup of negative charge at C3(PEP). The ensuing condensation of C3(PEP) with C1(A5P) would be favored by a proton transfer from the phosphate moiety of PEP to the aldehyde carbonyl of A5P to generate the hydroxyl. Overall, the process can be described as a syn addition of water and A5P to the si side of PEP.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: H S
authors.initials: HS
authors.lastname: Duewel
authors.forename: R W
authors.initials: RW
authors.lastname: Woodard
authors.forename: D L
authors.initials: DL
authors.lastname: Gatti
mesh_terms: D000446:Aldehyde-Lyases / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D010428:Pentosephosphates / Q000737:chemistry; D010728:Phosphoenolpyruvate / Q000737:chemistry*; D012267:Ribosemonophosphates / Q000737:chemistry*; D013379:Substrate Specificity; D014867:Water / Q000737:chemistry*
chemical_list: D004791:Enzyme Inhibitors; D010428:Pentosephosphates; D012267:Ribosemonophosphates; D014867:Water; C028055:arabinose 5-phosphate; C031626:ribose-5-phosphate; D010728:Phosphoenolpyruvate; C020011:2-dehydro-3-deoxyphosphooctonate aldolase; D000446:Aldehyde-Lyases