(E)-Enolbutyryl-UDP-N-acetylglucosamine as a Mechanistic Probe of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB)

UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-acetylmuramic acid in the presence of NADPH. The X-ra...

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Bibliographic Details
Published in:Biochemistry (Easton) 1996-02, Vol.35 (5), p.1342-1351
Main Authors: Lees, Watson J, Benson, Timothy E, Hogle, James M, Walsh, Christopher T
Format: Article
Language:English
Subjects:
Binding Sites
Carbohydrate Dehydrogenases - chemistry
Carbohydrate Dehydrogenases - metabolism
Crystallography, X-Ray
Deuterium Oxide - metabolism
Models, Chemical
Models, Molecular
Molecular Probes
NADP - metabolism
Oxidation-Reduction
Stereoisomerism
Uridine Diphosphate N-Acetylglucosamine - analogs & derivatives
Uridine Diphosphate N-Acetylglucosamine - chemistry
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Summary:UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-acetylmuramic acid in the presence of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc−MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc−MurB complex. In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product. This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-acetylmuramic acid 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 UDP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2−C3 single bond during catalysis.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi952287w