PDBsum entry 1x8f

Transferase

PDB id: 1x8f

Contents

Protein chain

272 a.a.

Waters ×46

References listed in PDB file

Key reference

Title

Crystal structures of escherichia coli kdo8p synthase complexes reveal the source of catalytic irreversibility.

Authors

R.Vainer, V.Belakhov, E.Rabkin, T.Baasov, N.Adir.

Ref.

J Mol Biol, 2005, 351, 641-652. [DOI no: 10.1016/j.jmb.2005.06.021]

PubMed id

16023668

Abstract

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 5-phosphate (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 visualize for the first time the role of His202 as an active-site gate. Examination of the crystal structures of KDO8PS with the KDO8P or 1dA5P shows these ligands bound to the enzyme in the PEP-binding sub-site, and not as expected to the A5P sub-site. Taken together, the structures presented here strengthen earlier evidence that this enzyme functions predominantly through positional catalysis, map out the roles of active-site residues and provide evidence that explains the total lack of catalytic reversibility.

Figure 2.
Figure 2. Electron density maps contoured over ligands bound within the KDO8PS active site. (a) The 2F[o] -F[c] electron density map overlaid onto the apo-KDO8PS His202 residue, contoured at 1.5s (orange) and at 0.9s (blue) showing the major and minor side-chain orientations. (b) The 2F[o] -F[c] electron density map overlaid onto the bound PEP contoured at 2s (orange) and at 1s (blue) is overlaid onto the PEP substrate. (c) The F[o] -F[c] omit electron density map overlaid onto the KDO8P product contoured at 2s (orange) and at 1s (blue). Note that the electron density allows the absolute assignment as the a-pyranoside anomer. (d) The F[o] -F[c] omit electron density map overlaid onto the 1dA5P analog contoured at 2s (orange) and at 1s (blue). In all Figures, atoms are colored according to: red, oxygen; blue, nitrogen; green, carbon; and magenta, phosphorus.

Figure 5.
Figure 5. Superposition of the KDO8PS binary structures with KDO8P (carbon atoms orange), 1dA5P (carbon atoms gray) and the mechanism-based inhibitor (Inh) as visualized in the 1G7V structure (carbon atoms magenta). The light cyan cartoon shows the position of the protein secondary structure surrounding the active site. Important residues have been placed to denote the direction of the active site. His202 (shown in ball-and-stick representation) is shown in its two alternative positions, with the closed conformation found in the KDO8PS·KDO8P structure denoted with an asterisk (*). The closed conformation is identical with the minor His202 orientation in the apo-KDO8PS structure as seen in Figure 1.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 351, 641-652) copyright 2005.