Publications

• A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes.

  Andersen G., Bjoernberg O., Polakova S., Pynyaha Y., Rasmussen A., Moeller K., Hofer A., Moritz T., Sandrini M.P., Merico A.M., Compagno C., Aekerlund H.E., Gojkovic Z., Piskur J.

  Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyve ... >> More

  Pyrimidine bases are the central precursors for RNA and DNA, and their intracellular pools are determined by de novo, salvage and catabolic pathways. In eukaryotes, degradation of uracil has been believed to proceed only via the reduction to dihydrouracil. Using a yeast model, Saccharomyces kluyveri, we show that during degradation, uracil is not reduced to dihydrouracil. Six loci, named URC1-6 (for uracil catabolism), are involved in the novel catabolic pathway. Four of them, URC3,5, URC6, and URC2 encode urea amidolyase, uracil phosphoribosyltransferase, and a putative transcription factor, respectively. The gene products of URC1 and URC4 are highly conserved proteins with so far unknown functions and they are present in a variety of prokaryotes and fungi. In bacteria and in some fungi, URC1 and URC4 are linked on the genome together with the gene for uracil phosphoribosyltransferase (URC6). Urc1p and Urc4p are therefore likely the core components of this novel biochemical pathway. A combination of genetic and analytical chemistry methods demonstrates that uridine monophosphate and urea are intermediates, and 3-hydroxypropionic acid, ammonia and carbon dioxide the final products of degradation. The URC pathway does not require the presence of an active respiratory chain and is therefore different from the oxidative and rut pathways described in prokaryotes, although the latter also gives 3-hydroxypropionic acid as the end product. The genes of the URC pathway are not homologous to any of the eukaryotic or prokaryotic genes involved in pyrimidine degradation described to date. << Less

  J. Mol. Biol. 380:656-666(2008) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enzymatic characterization of a prokaryotic urea carboxylase.

  Kanamori T., Kanou N., Atomi H., Imanaka T.

  We identified the first prokaryotic urea carboxylase (UCA) from a member of the alpha subclass of the class Proteobacteria, Oleomonas sagaranensis. This enzyme (O. sagaranensis Uca) was composed of 1,171 amino acids, and its N-terminal region resembled the biotin carboxylase domains of various bio ... >> More

  We identified the first prokaryotic urea carboxylase (UCA) from a member of the alpha subclass of the class Proteobacteria, Oleomonas sagaranensis. This enzyme (O. sagaranensis Uca) was composed of 1,171 amino acids, and its N-terminal region resembled the biotin carboxylase domains of various biotin-dependent carboxylases. The C-terminal region of the enzyme harbored the Met-Lys-Met motif found in biotin carboxyl carrier proteins. The primary structure of the enzyme was 45% identical to that of the urea carboxylase domain of urea amidolyase from Saccharomyces cerevisiae. O. sagaranensis Uca did not harbor the allophanate hydrolase domain found in the yeast enzyme, but a separate gene with structural similarity was found to be adjacent to the uca gene. Purified recombinant O. sagaranensis Uca displayed ATP-dependent carboxylase activity towards urea (V(max) = 21.2 micro mol mg(-1) min(-1)) but not towards acetyl coenzyme A (acetyl-CoA) and propionyl-CoA, indicating that the gene encoded a bona fide UCA and not an acetyl-CoA or propionyl-CoA carboxylase. The enzyme also exhibited high levels of activity towards acetamide and formamide. Kinetic parameters of the enzyme reaction were determined with ATP, urea, acetamide, and formamide. O. sagaranensis could grow on urea, acetamide, and formamide as sole nitrogen sources; moreover, ATP-dependent urea-degrading activity was found in cells grown with urea but not in cells grown with ammonia. The results suggest that the UCA of this organism may be involved in the assimilation of these compounds as nitrogen sources. Furthermore, orthologues of the O. sagaranensis uca gene were found to be widely distributed among Bacteria. This implies that there are two systems of urea degradation in Bacteria, a pathway catalyzed by the previously described ureases and the UCA-allophanate hydrolase pathway identified in this study. << Less

  J Bacteriol 186:2532-2539(2004) [PubMed] [EuropePMC]

• Urea carboxylase and allophanate hydrolase are components of a multifunctional protein in yeast.

  Sumrada R.A., Cooper T.G.

  Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci ... >> More

  Saccharomyces cerevisiae can use urea as sole nitrogen source by degrading it in two steps (urea carboxylase and allophanate hydrolase) to ammonia and carbon dioxide. We previously demonstrated that: 1) the enzymatic functions required for degradation are encoded in two tightly linked genetic loci and 2) pleiotropic mutations each resulting in the loss of both activities are found in both loci. These and other observations led to the hypothesis that urea degradation might be catalyzed by a multifunctional polypeptide. Waheed and Castric (1977) J. Biol. Chem. 252, 1628-1632), on the other hand, purified urea amidolyase from Candida utilis and reported it to be a tetramer composed of nonidentical 70- and 170-kilodalton subunits. To resolve the differing views of urea amidolyase structure, we purified the protein using rapid methods designed to avoid proteolytic cleavage. Application of these methods resulted in the isolation of a single, inducible and repressible, 204-kilodalton species. We observed no evidence for the existence of nonidentical subunits. A similar inducible, high molecular weight species was also detected in C. utilis. These biochemical results support our earlier hypothesis that urea degradation is carried out in yeast by an inducible and repressible protein composed of identical, multifunctional subunits. << Less

  J Biol Chem 257:9119-9127(1982) [PubMed] [EuropePMC]