A method for the enzymatic synthesis and HPLC purification of the peptidoglycan precursor UDP-N-acetylmuramic acid

Authors

  • Jon B Raymond,

    1. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 672, Rochester, NY 14642, USA
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  • Neil P Price,

    1. Departments of Anesthesiology and Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA
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  • Martin S Pavelka Jr.

    Corresponding author
    1. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Box 672, Rochester, NY 14642, USA
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*Corresponding author. Tel.: +1 (585) 275 4670; Fax: +1 (585) 473 9573, E-mail address: martin_pavelka@urmc.rochester.edu

Abstract

UDP-N-acetylmuramic acid (UDP-MurNAc) is a precursor for peptidoglycan biosynthesis in bacteria. A major difficulty in the study of this pathway is that UDP-MurNAc is not commercially available. We have developed an enzymatic synthesis scheme for UDP-MurNAc using two easily purified Escherichia coli polyhistidine tagged peptidoglycan biosynthesis enzymes, MurZ and MurB, followed by a single-step purification of UDP-MurNAc by high-performance liquid chromatography. The identity of the UDP-MurNAc synthesized by our method was confirmed by electrospray ionization mass spectrometry. Furthermore, we show that the UDP-MurNAc can support a UDP-MurNAc-l-alanine ligase reaction.

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