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Physiological consequences of thymidylate synthase ThyX utilization.

Abstract : The conversion of deoxyuridine 5'-monophosphate (dUMP) to deoxythymidine 5'-monophosphate (dTMP) by thymidylate synthases is one key step in the biosynthesis of pyrimidine deoxyribonucleotides. Two non homologous families of thymidylate synthases exist in the prokaryotic world (bacteria and archaea): ThyA and ThyX. The almost exclusive phylogenetic distribution of both enzyme families is complex and does not follow organismal phylogenetic relations. Proteins of both families do not share sequence or structural similarities, and the catalytic mechanisms and efficiencies are different. The work presented in this thesis demonstrates that ThyX activity is essential for the survival of the bacterium Rhodobacter capsulatus in absence of exogenous thymidine. Based upon the results of genetic complementation assays and a mathematical model of the folate metabolism, we showed that a very low dihydrofolate reductase activity is enough to allow survival of thyX containing organisms. Moreover, using a genetic approach and statistical analysis of more than 400 prokaryotic genomes, I was able to show that organisms using thymidylate synthase ThyX have slow DNA replication and growth rates, and mostly possess a small genome. Based upon these findings, I propose a model in which the weak activity of thymidylate synthases ThyX is limiting DNA replication and consequently genome expansion. Finally, I studied the pyrimidine metabolism in the human pathogenic bacterium Helicobacter pylori. In particular, I demonstrated that this bacterium is sensible to the anticancer drug ! 5-fluorouracil (5-FU) despite the absence of a uracil salvage pathway. I showed that the H. pylori orotate phosphoribosyltransferase is able to complement the uracil auxotrophy of an Escherichia coli strain, indicating that this enzyme could be responsible for the 5-FU sensitivity of H. pylori. The studies presented in this thesis allow a better understanding of the physiological consequences of the use of thymidylate synthase ThyX and provide an explanation for the apparent sporadic distribution of both thymidylate synthase families in the prokaryotic world.
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Frédéric Escartin. Physiological consequences of thymidylate synthase ThyX utilization.. Life Sciences [q-bio]. Ecole Polytechnique X, 2008. English. ⟨pastel-00004113⟩

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