Structures of Aquifex aeolicus KDO8P Synthase in Complex with R5P and PEP, and with a Bisubstrate Inhibitor:  Role of Active Site Water in Catalysis

• Published in: Biochemistry (Easton) Vol. 40; no. 51; pp. 15676 - 15683
• Main Authors: Wang, Jian, Duewel, Henry S, Woodard, Ronald W, Gatti, Domenico L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 25-12-2001
• Subjects: Aldehyde-Lyases - antagonists & inhibitors; Aldehyde-Lyases - chemistry; Binding Sites; Catalysis; Crystallography, X-Ray; Enzyme Inhibitors - chemistry; Gram-Negative Bacteria - enzymology; Hydrogen Bonding; Models, Molecular; Pentosephosphates - chemistry; Phosphoenolpyruvate - chemistry; Ribosemonophosphates - chemistry; Substrate Specificity; Water - chemistry
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi015568e

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 Cd2+ and located on the si side of PEP. This water molecule is no longer present because it cannot form a hydrogen bond with C2-OHR5P, which is oriented in a different direction from C2-OHA5P. 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 Cd2+. The current work supports a mechanism for the synthesis of KDO8P, in which a hydroxide ion on the si side of PEP attacks C2PEP, forming a tetrahedral-like intermediate with a buildup of negative charge at C3PEP. The ensuing condensation of C3PEP with C1A5P 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.