Abstract
Wall teichoic acids are a chemically diverse group of anionic polymers that constitute up to 50% of the Gram-positive cell wall. These polymers play a pivotal role in virulence and have been implicated in a diverse range of physiological functions. The TagF-like family of enzymes has been shown to be responsible for wall teichoic acid priming and polymerization events. Although many such enzymes are well validated therapeutic targets, a mechanistic understanding of this enzyme family has remained elusive. TagF is the prototypical teichoic acid polymerase and uses CDP-glycerol to catalyze synthesis of the linear (1,3)-linked poly(glycerol phosphate) teichoic acid in Bacillus subtilis 168. Here we used a synthetic soluble analog of the natural substrate of the enzyme, Lipid , to conduct the first detailed mechanistic investigation of teichoic acid polymerization. Through the use of a new high pressure liquid chromatography-based assay to monitor single glycerol phosphate incorporations into the Lipid analog, we conducted a detailed analysis of reaction product formation patterns and unequivocally showed TagF to be non-processive in vitro. Furthermore by monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same K(m) value of 2.6 microm and validated use of Bi Bi velocity expressions to model the TagF enzyme system. Initial rate analysis showed that TagF catalyzes a sequential Bi Bi mechanism where both substrates are added to the enzyme prior to product release consistent with a single displacement chemical mechanism.
Highlights
Wall teichoic acids are a group of phosphate-containing anionic carbohydrate polymers that constitute up to 50% of the dry weight of the Gram-positive cell wall [1]
By monitoring the kinetics of polymerization, we showed that Lipid analog species varying in size have the same Km value of 2.6 M and validated use of Bi Bi velocity expressions to model the TagF enzyme system
Our knowledge of wall teichoic acid synthesis largely stems from studies conducted in the model bacterium Bacillus subtilis 168, which expresses a linear [1,3]linked poly(glycerol phosphate) teichoic acid [5,6,7]
Summary
Shown is the nomenclature proposed for wall teichoic acid biosynthetic intermediates [17]. Intermediates were named according to the enzyme utilizing the molecule as a substrate. Lipid ␣ is the substrate for TagA, Lipid  is the substrate for TagB. Lipid .n species are substrates for TagF where n indicates the number of glycerol phosphate residues in the molecule. Lipid .1 is the product of the TagB-catalyzed priming reaction where a single glycerol phosphate residue is added. Lipid .1 is the product of the TagB-catalyzed priming reaction where a single glycerol phosphate residue is added. und, undecaprenol; P, phosphate; GroP, sn-glycerol-3-phosphate
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