| Abstract
| - UDP-N-acetylenolpyruvylglucosamine reductase(MurB), a peptidoglycan biosynthetic enzymefrom Escherichia coli, reduces both (E)- and(Z)-isomers of enolbutyryl-UDP-GlcNAc, C4analogs of thephysiological C3 enolpyruvyl substrate, toUDP-methyl-N-acetylmuramic acid in the presence ofNADPH.The X-ray crystal structure of the(E)-enolbutyryl-UDP-GlcNAc−MurB complex is similar to thatof theenolpyruvyl-UDP-GlcNAc−MurB complex. In both structures thegroups thought to be involved in hydridetransfer to C3 and protonation at C2 of the enol ether substrate arearranged anti relative to the enoldouble bond. The stereochemical outcome of reduction of(E)-enolbutyryl-UDP-GlcNAc by NADPD inD2O is thus predicted to yield a(2R,3R)-dideuterio product. This wasvalidated by conversion of the2,3-dideuterio-UDP-methyl-N-acetylmuramic acid product to2,3-dideuterio-2-hydroxybutyrate, which wasshown 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 topartitionbetween reduction to UDP-methyl-N-acetylmuramic andisomerization to the (Z)-substrate isomer intheMurB active site, indicative of a C2 carbanion/enol species that issufficiently long-lived to rotate aroundthe C2−C3 single bond during catalysis.
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