Biosynthesis of D-alanyl-lipoteichoic acid: characterization of ester-linked D-alanine in the in vitro-synthesized product

Abstract

d-Alanyl-lipoteichoic acid (d-alanyl-LTA) contains d-alanine ester residues which control the ability of this polyer to chelate Mg(2+). In Lactobacillus casei a two-step in vitro reaction sequence catalyzed by the d-alanine-activating enzyme and d-alanine:membrane acceptor ligase incorporates d-alanine into membrane acceptor. In this paper we provide additional evidence that the in vitro system catalyzes the covalent incorporation of d-[(14)C]alanine into membrane acceptor which is the poly([(3)H]glycerol phosphate) moiety of d-alanyl-LTA. This conclusion was supported by the observation that the d-[(14)C]alanine and [(3)H]glycerol labels of the partially purified product were co-precipitated by antiserum containing globulins specific for poly(glycerol phosphate). The isolation of d-[(14)C]alanyl-[(3)H]glycerol from d-[(14)C]alanine.[(3)H]glycerol-labeled d-alanyl-LTA synthesized in the in vitro system indicated that the d-alanine was linked to the poly(glycerol phosphate) chain of the LTA. A comparison of the reactivities of the d-alanine residues of d-alanyl-glycerol and d-alanyl-LTA supported the conclusion that the incorporated residue of d-alanine was attached by an ester linkage. Thus, the data indicated that the in vitro system catalyzes the incorporation of d-alanine covalently linked by ester linkages to the glycerol moieties of the poly(glycerol phosphate) chains of d-alanyl-LTA. New procedures are presented for the partial purification of d-alanyl-LTA with a high yield of ester-linked d-alanine and for the sequential degradation of the poly(glycerol phosphate) moiety substituted with d-alanine of d-alanyl-LTA with phosphodiesterase II/phosphatase from Aspergillus niger.