RESUMO
Most fungi cannot use pyrimidines or their degradation products as the sole nitrogen source. Previously, we screened several yeasts for their ability to catabolise pyrimidines. One of them, Saccharomyces kluyveri, was able to degrade the majority of pyrimidines. Here, a series of molecular techniques have been modified to clone pyrimidine catabolic genes, study their expression and purify the corresponding enzymes from this yeast. The pyd2-1 mutant, which lacked the 5,6-dihydropyrimidine amidohydrolase (DHPase) activity, was transformed with wild-type S. kluyveri genomic library. The complementing plasmid contained the full sequence of the PYD2 gene, which exhibited a high level of homology with mammalian DHPases and bacterial hydantoinases. The organisation of PYD2 showed a couple of specific features. The 542-codons open reading frame was interrupted by a 63 bp intron, which does not contain the Saccharomyces cerevisiae branch-point sequence, and the transcripts contained a long 5' untranslated leader with five or six AUG codons. The derived amino acid sequence showed similarities with dihydroorotases, allantoinases and uricases from various organisms. Surprisingly, the URA4 gene from S. cerevisiae, which encodes dihydroorotase, shows greater similarity to PYD2 and other catabolic enzymes than to dihydroorotases from several other non-fungal organisms. The S. kluyveri DHPase was purified to homogeneity and sequencing of the N-terminal region revealed that the purified enzyme corresponds to the PYD2 gene product. The enzyme is a tetramer, likely consisting of similar if not identical subunits each with a molecular mass of 59 kDa. The S. kluyveri DHPase was capable of catalysing both dihydrouracil and dihydrothymine degradation, presumably by the same reaction mechanism as that described for mammalian DHPase. On the other hand, the regulation of the yeast PYD2 gene and DHPase seem to be different from that in other organisms. DHPase activity and Northern analysis demonstrated that PYD2 expression is inducible by dihydrouracil, though not by uracil. Apparently, dihydrouracil and DHPase represent an important regulatory checkpoint of the pyrimidine catabolic pathway in S. kluyveri.
Assuntos
Amidoidrolases/genética , Amidoidrolases/metabolismo , Saccharomyces/enzimologia , Saccharomyces/genética , Regiões 5' não Traduzidas/genética , Amidoidrolases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Escherichia coli , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases. All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes.
Assuntos
Amidoidrolases/metabolismo , Amidoidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dictyostelium/enzimologia , Drosophila melanogaster/enzimologia , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Conformação Proteica , RNA Mensageiro/genética , Saccharomyces/enzimologia , Saccharomyces/genética , Saccharomyces/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosAssuntos
Purinas/metabolismo , Pirimidinas/metabolismo , Saccharomyces/crescimento & desenvolvimento , Meios de Cultura , Mutação , Saccharomyces/genética , Saccharomyces/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Especificidade da EspécieAssuntos
Drosophila melanogaster/metabolismo , Fluoruracila/farmacologia , Pirimidinas/metabolismo , Uracila/farmacologia , Alelos , Animais , Cromatografia Líquida de Alta Pressão , Cruzamentos Genéticos , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Feminino , Masculino , Mutagênese , Pirimidinas/isolamento & purificação , Especificidade da Espécie , Cromossomo XRESUMO
Transduced deoxyribonucleoside kinases (dNK) can be used to kill recipient cells in combination with nucleoside prodrugs. The Drosophila melanogaster multisubstrate dNK (Dm-dNK) displays a superior turnover rate and has a great plasticity regarding its substrates. We used directed evolution to create Dm-dNK mutants with increased specificity for several nucleoside analogs (NAs) used as anticancer or antiviral drugs. Four mutants were characterized for the ability to sensitize Escherichia coli toward analogs and for their substrate specificity and kinetic parameters. The mutants had a reduced ability to phosphorylate pyrimidines, while the ability to phosphorylate purine analogs was relatively similar to the wild-type enzyme. We selected two mutants, for expression in the osteosarcoma 143B, the glioblastoma U-87M-G and the breast cancer MCF7 cell lines. The sensitivities of the transduced cell lines in the presence of the NAs fludarabine (F-AraA), cladribine (CdA), vidarabine and cytarabine were compared to the parental cell lines. The sensitivity of 143B cells was increased by 470-fold in the presence of CdA and of U-87M-G cells by 435-fold in the presence of F-AraA. We also show that a choice of the selection and screening system plays a crucial role when optimizing suicide genes by directed evolution.
Assuntos
Antimetabólitos , Drosophila melanogaster/enzimologia , Terapia Genética/métodos , Mutação , Neoplasias/terapia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cladribina/uso terapêutico , Citarabina/uso terapêutico , Evolução Molecular Direcionada/métodos , Genes Transgênicos Suicidas , Glioblastoma/terapia , Humanos , Dose Letal Mediana , Osteossarcoma/terapia , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Purinas/metabolismo , Especificidade por Substrato , Transdução Genética/métodos , Vidarabina/análogos & derivados , Vidarabina/uso terapêuticoRESUMO
A Drosophila melanogaster mutant, fs(1)pyrSu(b), carrying a mutation that maps to the tip of the X chromosome, has been isolated. The mutation, when present alone, does not confer a detectable phenotype. However, this mutation causes female sterility and reduces embryonic viability when combined with mutations which deregulate the pyrimidine and beta-alanine pools. Embryos that are homozygous for the mutations fs(1)pyrSu(b), rSu(b) [previously designated as Su(b)] and b, and originate from a female parent homozygous for the three mutations show severely reduced viability. Newly laid eggs begin development normally, but the majority of the embryos die just before the eggs are due to hatch.
Assuntos
Drosophila melanogaster/genética , Genes de Insetos , Mutação , Alelos , Animais , Cruzamentos Genéticos , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Feminino , Genes Letais , Genes Recessivos , Heterozigoto , Homozigoto , Infertilidade Feminina/genética , Masculino , Fenótipo , Pirimidinas/metabolismo , beta-Alanina/metabolismoRESUMO
The six biochemical steps of the de novo pyrimidine biosynthesis pathway are conserved in all known organisms. However, in animals and fungi, unlike prokaryotes, at least the first two activities are grouped on a multifunctional enzyme. Here, we report cloning, mapping and transcriptional characterization of some pyrimidine biosynthesis genes in the filamentous fungus Aspergillus nidulans. The first two steps of the pathway are performed by a multifunctional enzyme comprising the activities of carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). This polypeptide is encoded by a 7 kbp cluster gene, pyrABCN, which has a high degree of nucleotide identity with the Ura2 gene in Saccharomyces cerevisiae. The enzyme of the third step, dihydroorotase (DHOase), is encoded by a separate locus, pyrD. However, the pyrABCN gene apparently contains an evolutionary remnant of a DHOase-encoding sequence, similarly to the Ura2 gene of Saccharomyces cerevisiae. The pyrABCN gene is transcribed as a single 7 kb mRNA species. The level of transcripts of pyrABCN, pyrD and, to a lesser degree, pyrF genes responds to the presence of exogenous pyrimidines and to the conditions of pyrimidine starvation. Derepression of pyrABCN and pyrD under pyrimidine starvation is noticeably enhanced in pyrE mutants that accumulate dihydroorotic acid. The pyrABCN gene maps to the distal portion of the right arm of the chromosome VIII, whereas the pyrD gene, in contrast to early genetic data, is closely linked to the brlA gene and located to the right of it. Our data on mitotic recombination should help to verify the genetic map of the chromosome VIII. Comparison of amino acid sequences of active dihydroorotases with related enzymes and with their non-functional homologues in yeast and Aspergillus indicates that the active dihydroorotases from fungi are more similar to ureases and enzymes of the pyrimidine degradation pathway. The 'silent' dihydroorotase domains of the multifunctional enzymes from fungi and active DHOase domains of the multifunctional enzymes in higher eukaryotes are more closely related to bacterial dehydroorotases.
Assuntos
Aspartato Carbamoiltransferase/genética , Aspergillus nidulans/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Di-Hidro-Orotase/genética , Genes Fúngicos , Complexos Multienzimáticos/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Filogenia , Pirimidinas/biossíntese , RNA Mensageiro/análise , Mapeamento por Restrição , Alinhamento de Sequência , Transcrição GênicaRESUMO
The ability to propagate under anaerobic conditions is an essential and unique trait of brewer's or baker's yeast ( Saccharomyces cervisiae). To understand the evolution of facultative anaerobiosis we studied the dependence of de novo pyrimidine biosynthesis, more precisely the fourth enzymic activity catalysed by dihydroorotate dehydrogenase (DHODase), on the enzymes of the respiratory chain in several yeast species. While the majority of yeasts possess a mitochondrial DHODase, Saccharomyces cerevisiae has a cytoplasmatic enzyme, whose activity is independent of the presence of oxygen. From the phylogenetic point of view, this enzyme is closely related to a bacterial DHODase from Lactococcus lactis. Here we show that S. kluyveri, which separated from the S. cerevisiae lineage more than 100 million years ago, represents an evolutionary intermediate, having both cytoplasmic and mitochondrial DHODases. We show that these two S. kluyveri enzymes, and their coding genes, differ in their dependence on the presence of oxygen. Only the cytoplasmic DHODase promotes growth in the absence of oxygen. Apparently a Saccharomyces yeast progenitor which had a eukaryotic-like mitochondrial DHODase acquired a bacterial gene for DHODase, which subsequently allowed cell growth gradually to become independent of oxygen.