d-Alanine: Membrane Acceptor Ligase from Lactobacillus casei

The enzymatic incorporation of d-alanine into membranes of Lactobacillus casei (ATCC 7469) is described. The activity is dependent on ATP, supernatant fraction and membrane fragments; it is enhanced by the addition of Mg2+. The incorporation is insensitive to ribonuclease. The product of this reaction is characterized as a hydroxylamine (1 m, pH 7.0, 37°) labile ester that is not extracted into lipophilic solvents. The lability of the incorporated d-alanine in hydroxylamine is similar to that observed for the d-alanine ester residues in the glycerol teichoic acid from this organism. The chromatographic properties of the product are not consistent with alanyl phosphatidylglycerol. The Km for d-alanine and d-α-NH2-n-butyric acid is 18 µm and 850 µm, respectively. l-[14C]Alanine is not incorporated. The following analogues are effective inhibitors of d-alanine incorporation: (a) d-α-NH2-n-butyric acid; (b) dl-alanine hydroxamate; (c) dl-α-amino-n-butyric acid hydroxamate; (d) dl-alanine amide; and (e) d-serine. Although the specificity profile is similar to that described for the d-alanine-activating enzyme (Baddiley, J., and Neuhaus, F. C., Biochem. J., 75, 579 (1960)), the supernatant factor was partially separated from this enzyme and alanine racemase by gel filtration on Sephadex G-150. It is proposed that the supernatant fraction contains an enzyme, d-alanine:membrane acceptor ligase, that is involved in the introduction of d-alanine ester residues into membranes of this organism.