Overexpression, purification, and characterization of UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli.
UDP-N-acetylmuramyl:L-alanine ligase from Escherichia coli was overexpressed more than 600-fold and purified to near homogeneity. The purified enzyme was found to ligate L-alanine, L-serine, and glycine, as well as the nonnatural amino acid beta-chloro-L-alanine, to UDP-N-acetylmuramic acid. On the basis of (i) the specificity constants of the enzyme determined for L-alanine, L-serine, and glycine and (ii) the levels of these amino acids in the intracellular pool, it was calculated that the rates of incorporation of L-serine and glycine into peptidoglycan precursor metabolites could maximally amount to 0.1 and 0.5%, respectively, of the rate of L-alanine incorporation.
Department of Infectious Diseases, F. Hoffman-La Roche AG, CH-4002 Basel, Switzerland.
M
M
Gubler
Y
Y
Appoldt
W
W
Keck
D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008025:Ligases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D014538:Uridine Diphosphate N-Acetylmuramic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
D014538:Uridine Diphosphate N-Acetylmuramic Acid; C076606:UDP-N-acetylmuramylalanine; D008025:Ligases