(E)-ENOLBUTYRYL-UDP-N-ACETYLGLUCOSAMINE AS A MECHANISTIC PROBE OF UDP-N-ACETYLENOLPYRUVYLGLUCOSAMINE REDUCTASE (MURB)

UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan b iosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-is omers of enolbutyryl-UDP-GlcNAc, C-4 analogs of the physiological C-3 enolpyruvyl substrate, to UDP-methyl-N-acetylmuramic acid in the prese nce of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-G lcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-Mu rB complex. In both structures the groups thought to be involved in hy dride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemica l outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O i s thus predicted to yield a (2R,3R)-dideuterio product, This was valid ated by conversion of the 2,3-dideuterio-UDP-methyl-N-acetylmuramic ac id product to 2,3-dideuterio-2-hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R ,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate. Remarkably, the (E )-enolbutyryl-UDP-GlcNAc was found to partition between reduction to U DP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isome r in the MurB active site, indicative of a C2 carbanion/enol species t hat is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis.