Probing the Stereochemistry of E. coli 3-Deoxy-d-arabino-heptulosonate 7-Phosphate Synthase (Phenylalanine-Sensitive)-Catalyzed Synthesis of KDO 8-P Analogues

The five-carbon phosphorylated monosaccharide analogues, d-arabinose 5-phosphate, d-ribose 5-phosphate, and 2-deoxy-d-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-2H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-2H]-PEP (3S)-[3-2H]-3-deoxy-d-manno-octulosonate 8-phosphate, (3S)-[3-2H]-3-deoxy-d-altro-octulosonate 8-phosphate, and (3S)-[3-2H]-3,5-dideoxy-d-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-2H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates d-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by 1H and 19F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.