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It is generally believed that pyrimidine degradation is initiated by either a reductive or an oxidative step! Are there really no alternatives? Degradation of pyrimidines is of a great importance in humans. Defects in the genes involved in the corresponding pathway cause severe symptoms, and especially in cancer patients a combination of gene defects and chemotherapy with e.g. 5-fluorouracil (a widely used anti-cancer drug) can have fatal consequences. The first step in the catabolic pathway performed by dihydropyrimidine dehydrogenase (DHPDH), has been found in mammals, insects, plants and bacteria. This first step has so far not been found in the fungi kingdom, but two genes (PYD2 and PYD3) encoding the two subsequent steps in the catabolic pathway, have previously been characterized in the yeast, Saccharomyces kluyveri. In this thesis, the origin of the uracil degradation pathway in yeast, and the genetic background for uracil and beta-alanine (BAL) catabolism in S. kluyveri, were investigated. The ability to use uracil, dihydrouracil (DHU), beta-ureidopropionate (BUP) and BAL as nitrogen source was studied in thirty-eight yeast species selected to cover the ”Saccharomyces complex”, which originated more than 200 mill. years ago. Uracil, DHU and BUP degradation were found to be linked in almost all the thirty-eight yeast species tested, and the pathway was apparently lost after the whole genome duplication. The ability to use BAL as sole nitrogen source was not linked to the three others, and was lost more or less randomly. In order to study the genetic background of uracil degradation in S. kluyveri a number of mutants defective in uracil degradation were isolated, and the defects were found to belong to six loci (PYD11,12,13,14,15,16). None of these loci were allelic to the previously described PYD2 and PYD3 loci, and all of the mutants could use DHU and BUP as sole nitrogen source. Targeted disruption of all eight loci showed that uracil is degraded by a pathway consisting of the PYD1X genes, while DHU is degraded by a pathway consisting of PYD2 and PYD3. Surprisingly, uracil is degraded via UMP, and urea is an intermediate. A new gene, termed PYD4, found to be involved in DHU degradation was isolated. PYD4 encodes a pyridoxal-5’-phosphate-dependent aminotransferase, which shows similar activity and substrate specificity as mammalian BAL/gamma-aminobutyrate aminotransferase (BAL/GABA-AT) [EC 188.8.131.52]. S. kluyveri also has an UGA1 encoded GABA-AT [EC 184.108.40.206], which is specific for GABA. The original gene was apparently duplicated, thus giving PYD4 and UGA1, less than 200 mill. years ago.
|Place of Publication||Kgs. Lyngby|
|Publisher||Technical University of Denmark|
|Number of pages||155|
|Publication status||Published - Sep 2006|
Structure-Function Relationship and Evolution of Enzymes Involved in Degradation of Anti-Cancer Drugs
Andersen, G., Piskur, J., Jørck-Ramberg, D., Martinussen, J., Bianchi, V. & Holmberg, S.
01/10/2002 → 15/09/2006