The study of many of glycosyltransferases is limited due to an inadequate access to sugar
nucleotides. Preparation of sugar nucleotides through the use of nucleotidylyltransferases
with broad substrate specificities is gaining significant interest and offers high yields and
stereospecificity. Physiologically, the glucose 1-phosphate thymidylyltransferase
catalyzes the condensation of ?-D-glucose 1-phosphate and deoxythymidine triphosphate
to yield deoxythymidine diphospho glucose. Exploiting and targeting these enzymes also
has the potential of yielding new therapeutics.
Cps2L is a thymidylyltransferase isolated from Streptococcus pneumoniae, with
broad substrate flexibility. The substrate specificity of Cps2L was evaluated with new
sugar 1-phosphate analogues to gain further insight into substrate and inhibitor
requirements. Several sugar 1-phosphate analogues including sugar 1C-phosphonates
(and analogues thereof), 2-deoxy-2-fluorosugar 1-phosphates, and glucopyranose 1-
boranophosphates have been used to probe the sugar 1-phosphate modification tolerance
of Cps2L. In addition, NMR spectroscopy was used to determine the anomeric
stereochemistry of 2-deoxy-2-fluorosugars nucleotide products. For those substrates that
were accepted by Cps2L, steady-state kinetic parameters were determined. The enzyme
is able to almost equally form Michaelis complexes with different sugar substrates,
whereas the turnover values for obtaining the corresponding sugar nucleotide were
different. The evaluation of the substrate tolerance of Cps2L, as well as the synthesis of
?-D-glucose-1C-thiophosphonate, a difluoro and a bisphosphono analogue of ?-D-glucose
1C-phosphonate will be described.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:NSHD.ca#10222/12815 |
| Date | 22 April 2010 |
| Creators | Beaton, Stephen A. |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_US |
| Detected Language | English |
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