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 B-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-H-2]-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-H-2]-PEP (3S)-[3-H-2]-3-deoxy-D-manno-octulosonate S-phosphate, (3S)-[3-H-2]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-H-2]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3(2)H]-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 H-1 and F-19 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.