The proposed mechanistic pathway for the reaction catalyzed by 3-deoxy-D-manno-2-octulosonate-8-phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate. Two 2-deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring-opening and possessing the stereochemistry of either the alpha-pyranase (compound 1) or the beta-pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 microM and 303 microM, respectively. Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki = 590 microM) suggests that both the alpha- and the beta-pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the beta-anomer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable. More importantly, however, the isosteric phosphonate analogue 2,6-anhydro-3-deoxy-2 beta-phosphonylmethyl-8-phosphate-D-glycero-D-talo-octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki value of 5 microM. Two hitherto unrecognized aspects of the mechanism of the synthase were identified. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open-chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open-chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme. Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenolpyruvate to the re face of the carbonyl of arabinose 5-phosphate, the present observation involving analogue 3 suggest that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the beta-configuration.