Crystal structures of Escherichia coli KDO8P synthase complexes reveal the source of catalytic irreversibility.

The enzyme 3-deoxy- D-manno-2-octulosonate-8-phosphate synthase (KDO8PS) catalyses the condensation of arabinose 5-phosphate (A5P) and phosphoenol pyruvate (PEP) to obtain 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P). We have elucidated initial modes of ligand binding in KDO8PS binary complexes by X-ray crystallography. Structures of the apo-enzyme and of binary complexes with the substrate PEP, the product KDO8P and the catalytically inactive 1-deoxy analog of arabinose 5phosphate (1dA5P) were obtained. The KDO8PS active site resembles an irregular funnel with positive electrostatic potential situated at the bottom of the PEP-binding sub-site, which is the primary attractive force towards negatively charged phosphate moieties of all ligands. The structures of the ligand-free apo-KDO8PS and the binary complex with the product KDO8P visualizeforthefirsttimetheroleofHis202asanactive-sitegate.Examination of the crystal structures of KDO8PS with the KDO8P or 1dA5P shows these ligandsboundtotheenzymeinthePEP-bindingsub-site,andnotasexpected to the A5P sub-site. Taken together, the structures presented here strengthen earlierevidencethatthisenzymefunctionspredominantlythroughpositional catalysis, map out the roles of active-site residues and provide evidence that explains the total lack of catalytic reversibility. q 2005 Elsevier Ltd. All rights reserved.