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Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase.
Biochemistry. 1993 Mar 02; 32(8):2024-30.B

Abstract

The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed.

Authors+Show Affiliations

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.No affiliation info availableNo 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, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8448160

Citation

Benson, T E., et al. "Overexpression, Purification, and Mechanistic Study of UDP-N-acetylenolpyruvylglucosamine Reductase." Biochemistry, vol. 32, no. 8, 1993, pp. 2024-30.
Benson TE, Marquardt JL, Marquardt AC, et al. Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry. 1993;32(8):2024-30.
Benson, T. E., Marquardt, J. L., Marquardt, A. C., Etzkorn, F. A., & Walsh, C. T. (1993). Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase. Biochemistry, 32(8), 2024-30.
Benson TE, et al. Overexpression, Purification, and Mechanistic Study of UDP-N-acetylenolpyruvylglucosamine Reductase. Biochemistry. 1993 Mar 2;32(8):2024-30. PubMed PMID: 8448160.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Overexpression, purification, and mechanistic study of UDP-N-acetylenolpyruvylglucosamine reductase. AU - Benson,T E, AU - Marquardt,J L, AU - Marquardt,A C, AU - Etzkorn,F A, AU - Walsh,C T, PY - 1993/3/2/pubmed PY - 1993/3/2/medline PY - 1993/3/2/entrez SP - 2024 EP - 30 JF - Biochemistry JO - Biochemistry VL - 32 IS - 8 N2 - The recently isolated Escherichia coli murB gene (Pucci et al., 1992) has been cloned into an expression vector and the encoded UDP-N-acetylenolpyruvylglucosamine reductase (EC 1.1.1.158) was overproduced to about 10% of soluble cell protein. The encoded 38-kDa protein has been purified to near homogeneity. It was found to be a monomer and to contain stoichiometric amounts of bound FAD which is reducible in catalytic turnover. The enzyme utilizes the 4-pro-S hydrogen of NADPH to reduce the enolpyruvyl group of UDP-N-acetylglucosamine enolpyruvate to the lactyl ether in UDP-N-acetylmuramic acid. NMR analysis of products from 2H2O and 4S-[2H]NADPH incubations establishes that a hydride from NADPH via E.FADH2 is transferred to the beta-methyl of the 3-O-lactyl moiety and a proton from solvent to the alpha-carbon of the lactyl moiety of UDP-N-acetylmuramic acid. A mechanism for this unusual enolether reduction in bacterial cell wall assembly is proposed. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8448160/Overexpression_purification_and_mechanistic_study_of_UDP_N_acetylenolpyruvylglucosamine_reductase_ L2 - https://ecocyc.org/gene?orgid=ECOLI&id=EG11205 DB - PRIME DP - Unbound Medicine ER -