Stereochemistry and mechanism of the GDP-mannose dehydratase reaction

The reaction catalyzed by bacterial GDP-mannose dehydratase (E.C. 4.2.1.47), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose (GDP-6-deoxy-D-lyxo-hexos-4-ulose), was studied with (6R)- and (6S)-GDP-D-[4-2H1,6-3H]mannose. Conversion of these stereospecifically labeled substrates in the presence of excess unlabeled GDP-mannose into the 4-keto-6-deoxy derivatives followed by Kuhn-Roth oxidation gave acetic acid samples which were subjected to configurational analysis of the isotopically chiral methyl group. The observed F values of 64 for the material from the (6S) substrate and 31 for that from the (6R) isomer, corresponding to 48% e.e. R and 66% e.e. S configuration, respectively, of the methyl group indicate that (a) the oxidoreductase reaction involves transfer of H-4 to C-6, (b) the transfer is predominantly intramolecular, and (c) the transfer is stereospecific, H-4 replacing the C-6 hydroxyl group with inversion of configuration. A mechanism for the reaction is proposed on the basis of these results.