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 .