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 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.
Raymond
Jon B
JB
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Box 672, Rochester, NY 14642, USA.
Price
Neil P
NP
Pavelka
Martin S
MS
D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D002237:Carbohydrate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D010457:Peptidoglycan / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000302:isolation & purification*
D010457:Peptidoglycan; D011994:Recombinant Proteins; D014538:Uridine Diphosphate N-Acetylmuramic Acid; C076606:UDP-N-acetylmuramylalanine; C033223:polyhistidine; D006639:Histidine; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase; D010453:Peptide Synthases; C023229:UDP-N-acetylmuramoyl-alanine synthetase