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

Abstract

D-Alanyl-lipoteichoic acid (D-ananyl-LTA) contains D-alanine ester residues which control the ability of this polymer to chelate Mg2+. In L. 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-[14C]alanine into membrane acceptor which is the poly([3H]glycerol phosphate) moiety of D-alanyl-LTA. This conclusion was supported by the observation that the D-[14C]alanine and [3H]glycerol labels of the partially purified product were co-precipitated by antiserum containing globulins specific for poly(glycerol phosphate). The isolation of D-[14C]alanyl-[3H]glycerol from D-[14C]alanine-[3H]glycerol-labeled D-alanyl-LTA synthesized in the 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.