The biosynthetic enzymes involved in wall teichoic acid biogenesis in Grampositive
bacteria have been the subject of renewed investigation in recent years with the
benefit of modem tools of biochemistry and genetics. Nevertheless, there have been only
limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old
experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage
resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the
wall teichoic acid glycosyltransferase. This study and others have provided only indirect
evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we
showed that deletion of tagE results in the loss of a-glucose at the C-2 position of
glycerol in the poly(glycerol phosphate) polymer backbone. We also report the first
kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using
clean synthetic substrates. We investigated the substrate specificity ofTagE using a wide
variety of acceptor substrates and showed that this enzyme has a strong kinetic preference
for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form.
Further, we showed that the enzyme recognizes its polymeric (and repetitive) substrate
with a sequential kinetic mechanism. This work provides direct evidence that TagE is the
wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for
further studies on the mechanism of wall teichoic acid glycosylation, a largely uncharted
aspect of wall teichoic acid biogenesis. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21158 |
| Date | 04 1900 |
| Creators | Allison, Sarah |
| Contributors | Brown, Eric, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
Page generated in 0.0023 seconds