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

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 col...

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Veröffentlicht in:	FEMS microbiology letters 2003-12, Vol.229 (1), p.83-89
Hauptverfasser:	Raymond, Jon B, Price, Neil P, Pavelka, Martin S
Format:	Artikel
Sprache:	eng
Schlagworte:	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
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Zusammenfassung:	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.
ISSN:	0378-1097 1574-6968
DOI:	10.1016/S0378-1097(03)00793-6