Insights into the mechanism of the teichoic acid polymerase from Bacillus subtilis and related enzymes

Teichoic acids (TA) are phosphate rich polymers which make up a significant proportion of the Gram‐positive bacterial cell wall. Despite their abundance, little is known regarding their function or biosynthesis. In addition, the teichoic acid biosynthetic pathway is comprised of several indispensable genes whose products represent potential targets for novel antibiotics. The TagF protein from Bacillus subtilis 168 is the poly(glycerol phosphate) polymerase responsible for the synthesis of wall TA and is the prototype member of a poorly understood family of similar enzymes. TagF shares no significant homology with any protein of known function. Alignment of its amino acid sequence with predicted TA synthetic enzymes from other organisms identified highly conserved regions specific to the TagF‐like family. Structural experiments localized the active site of TagF to its carboxyl‐terminus, consistent with bioinformatic predictions. pH analysis combined with site‐directed mutagenesis and in vivo complementation experiments identified two conserved histidine residues which are critical to TagF function. H474A and H612A variants showed a 5000‐fold reduction in k cat /K m while variants in four other conserved acidic residues showed minimal loss of activity. We extended these results to suggest mechanistic commonality across the TagF‐like family by demonstrating that analogous substitutions also impacted the activity of the homologous TagB protein. Our experiments give the first insights into how the members of this mysterious family of glycerophosphotransferases promote catalysis. This work was funded by the CIHR.