Andersen, Gorm

Det første skridt i nedbrydningen af pyrimidiner er enten en reduktiv eller en oxidativ reaktion! Er der virkelig ingen alternativer? Nedbrydningen af pyrimidiner har stor betydning i mennesket. Genetiske defekter i den tilhørende pathway medfører alvorlige symptomer og specielt i for kræftpatienter, som får pyrimidinbaseret kemoterapi (f.eks. 5-fluorouracil, som er et meget anvendt kræfthæmmende stof), kan disse defekter få letale konsekvenser. The første trin i den katabolske pathway udført af dihydropyrimidine dehydrogenase (DHPDH), er blevet fundet i pattedyr, insekter, planter og bakterier. Dette første skridt er dog endnu ikke blevet fundet i svampe riget, men to gener (PYD2 og PYD3), som koder for de efterfølgende trin i den katabolske pathway, er tidligere blevet karakteriseret i gæren, Saccharomyces kluyveri. I denne afhandling, bliver oprindelsen af uracil nedbrydningspathway’en i gær og de genetiske forudsætninger for uracil og beta-alanine (BAL) katabolisme i S. kluyveri undersøgt. Evnen til at bruge uracil, dihydrouracil (DHU), beta-ureidopropionate (BUP) og BAL som nitrogenkilde blev studeret i 38 gær arter. Disse var udvalgt, så de dækkede “Saccharomyces komplekset”, som opstod for mere end 200 mill. år siden. Evnen til at nedbryde uracil, DHU og BUP var sammenkædet i næsten alle 38 gær arter, som blev testet, og pathway’en blev tilsyneladende tabt efter genom duplikationen. Evnen til at bruge BAL som eneste nitrogenkilde var uafhængig af de tre andre, og blev tabt mere eller mindre tilfædigt. For at studere de genetiske forudsætninger for nedbrydning af uracil i S. kluyveri, blev et antal mutanter, som var defekte i nedbrydningen af uracil, isoleret, og defekterne tilhørte seks loci (PYD11,12,13,14,15,16). Ingen af disse var identiske med de to tidligere beskrevne, PYD2 og PYD3 loci og alle mutanterne kunne bruge DHU samt BUP som eneste nitrogenkilde. Målrettet ødelæggelse af alle otte loci viste, at uracil bliver nedbrudt via en pathway bestående af PYD1X generne, mens DHU bliver nedbrudt via en pathway bestånde af PYD2 og PYD3. Overraskende nok, så blev uracil nedbrudt via UMP og urea var et intermediat. Et nyt gen, kaldet PYD4, som er involveret i nedbrydningen af DHU, blev isoleret. PYD4 koder for en pyridoxal-5’-fosfat-afhængig aminotransferase, som har sammenlignelig aktivitet og substratspecificitet som BAL/gamma-aminobutyrate aminotransferase (BAL/GABA-AT) [EC 2.6.1.19] fra pattedyr. S. kluyveri har også en UGA1 kodet GABA-AT [EC 2.6.1.19], som er specifik for GABA. Det oprindelige gen blev tilsyneladende duplikeret for mindre end 200 ,mill. år siden, hvilket resulterede i PYD4 og UGA1.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 2.6.1.19]. S. kluyveri also has an UGA1 encoded GABA-AT [EC 2.6.1.19], which is specific for GABA. The original gene was apparently duplicated, thus giving PYD4 and UGA1, less than 200 mill. years ago

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Uracil and beta-alanine degradation in Saccharomyces Kluyveri - discovery of a novel catabolic pathway

http://orbit.dtu.dk/ws/files/5094539/thesis%20-%20gorm%20andersen.pdf

Uracil and beta-alanine degradation in Saccharomyces Kluyveri - discovery of a novel catabolic pathway

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