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(E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB).
Biochemistry. 1996 Feb 06; 35(5):1342-51.B

Abstract

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.

Authors+Show Affiliations

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8634262

Citation

Lees, W J., et al. "(E)-enolbutyryl-UDP-N-acetylglucosamine as a Mechanistic Probe of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB)." Biochemistry, vol. 35, no. 5, 1996, pp. 1342-51.
Lees WJ, Benson TE, Hogle JM, et al. (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Biochemistry. 1996;35(5):1342-51.
Lees, W. J., Benson, T. E., Hogle, J. M., & Walsh, C. T. (1996). (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). Biochemistry, 35(5), 1342-51.
Lees WJ, et al. (E)-enolbutyryl-UDP-N-acetylglucosamine as a Mechanistic Probe of UDP-N-acetylenolpyruvylglucosamine Reductase (MurB). Biochemistry. 1996 Feb 6;35(5):1342-51. PubMed PMID: 8634262.
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TY - JOUR T1 - (E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB). AU - Lees,W J, AU - Benson,T E, AU - Hogle,J M, AU - Walsh,C T, PY - 1996/2/6/pubmed PY - 1996/2/6/medline PY - 1996/2/6/entrez SP - 1342 EP - 51 JF - Biochemistry JO - Biochemistry VL - 35 IS - 5 N2 - 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. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8634262/_E__enolbutyryl_UDP_N_acetylglucosamine_as_a_mechanistic_probe_of_UDP_N_acetylenolpyruvylglucosamine_reductase__MurB__ L2 - https://doi.org/10.1021/bi952287w DB - PRIME DP - Unbound Medicine ER -