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Structures of Aquifex aeolicus KDO8P Synthase in Complex with R5P and PEP, and with a Bisubstrate Inhibitor: Role of Active Site Water in Catalysis
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 intermedi...
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Published in: | Biochemistry (Easton) 2001-12, Vol.40 (51), p.15676-15683 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | 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. |
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ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi015568e |