N‐Acetyimuramoyl‐l‐alkaline Amidase of Escherichia coli K12

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Abstract
Various experiments were carried out in an attempt to determine the possible physiological function of the N-acetylmuramoyl-L-alanine amidase purified from E. coli K12 on the basis of its activity on N-acetylmuramoyl-L-alanyl-D-.gamma.-glutamyl-meso-diaminopimelic acid [MurNAc-L-Ala-DGlu(msA2pm)]. A Km value of 0.04 mM was determined with this substrate. Specificity studies revealed that compounds with a MurNAc-L-Ala linkage are the most probable substrates of this enzyme in vivo. Purified amidase had no effect on purified peptidoglycan and only low levels (1-25%) of cleaved MurNAc-L-Ala linkages were detected in peptidoglycan isolated from normally growing cells. The action of the amidase in vivo on peptidoglycan was clearly detectable during autolysis. The amidase activity of cells treated by osmotic shock, ether or toluene, and that of mutants with altered outer membrane composition was investigated. Attempts to reveal a transfer reaction catalyzed by amidase were unsuccessful. By its location and specificity, amidase was clearly not involved in the formation of UDP-MurNAc. The possibility that it might be functioning in vivo as a hydrolase degrading exogeneous peptidoglycan fragments in the periplasma was substantiated by the fact that MurNAc itself and MurNAc-peptides could sustain growth of E. coli as sole C an N sources. Out of 200 thermosensitive mutants examined for altered amidase activity, only 2 strains has < 50% of the normal level of activity, whereas 10 possess > 50%. Two of the overproducers encountered presented a 4- to 5-fold increase in activity.