The wall teichoic acid polymerase TagF is non-processive in vitro and amenable to study using steady state kinetic analysis.
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.
Sewell
Edward W C
EW
Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8N 3Z5, Canada.
Pereira
Mark P
MP
Brown
Eric D
ED
D001412:Bacillus subtilis / Q000201:enzymology* / Q000378:metabolism; D002384:Catalysis; D002473:Cell Wall / Q000201:enzymology; D003565:Cytidine Diphosphate / Q000737:chemistry; D011756:Diphosphates / Q000737:chemistry; D005990:Glycerol / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008954:Models, Biological; D008956:Models, Chemical; D010710:Phosphates / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D013682:Teichoic Acids / Q000378:metabolism*; D013997:Time Factors; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism / Q000502:physiology*
D011756:Diphosphates; D008055:Lipids; D010710:Phosphates; D011108:Polymers; D013682:Teichoic Acids; D003565:Cytidine Diphosphate; D017855:Transferases (Other Substituted Phosphate Groups); C072175:CDP glycerol glycerophosphotransferase; D005990:Glycerol