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

• Published in: FEMS microbiology letters 2003-12, Vol.229 (1), p.83-89
• Main Authors: Raymond, Jon B, Price, Neil P, Pavelka, Martin S
• Format: Article
• Language: English
• Subjects: Action of physical and chemical agents; Alkyl and Aryl Transferases - genetics; Alkyl and Aryl Transferases - metabolism; Biological and medical sciences; Carbohydrate Dehydrogenases - genetics; Carbohydrate Dehydrogenases - metabolism; Chromatography, High Pressure Liquid - methods; Escherichia coli; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Histidine - genetics; Microbiology; Mycological methods and techniques used in mycology; Mycology; Peptide Synthases - genetics; Peptide Synthases - metabolism; Peptidoglycan; Peptidoglycan - biosynthesis; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; UDP- N-acetylmuramic acid; Uridine Diphosphate N-Acetylmuramic Acid - analogs & derivatives; Uridine Diphosphate N-Acetylmuramic Acid - biosynthesis; Uridine Diphosphate N-Acetylmuramic Acid - isolation & purification; Virology
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/S0378-1097(03)00793-6

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.