Reconstituting poly(glycerol phosphate) wall teichoic acid biosynthesis in vitro using authentic substrates

Wall teichoic acids (WTAs) are phosphate-rich anionic polymers that constitute a substantial portion of the Gram-positive cell wall. Recent work has demonstrated the importance of WTAs in cell shape, virulence and antibiotic resistance. These findings highlight WTA biosynthetic enzymes as attractive targets for novel antimicrobial agents. Due to challenges involved in the isolation of natural substrates, in vitro studies of the recombinant enzymes have largely employed soluble substrate analogues. Herein we present a semisynthetic approach to obtain the authentic precursor for WTA biosynthesis, Lipid alpha, complete with its polyisoprenoid lipid moiety. We show that this material can be used to reconstitute the activities of four enzymes involved in poly(glycerol phosphate) WTA biosynthesis in a detergent micelle. This work enables the creation of chemically defined and realistic systems for the study of interfacial catalysis by WTA biosyntheticmachinery, which could aid efforts to discover and develop novel agents against WTA biosynthesis.