Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

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.     

Saccharomyces kluyveri as a model organism to study pyrimidine degradation

The yeast Saccharomyces kluyveri (Lachancea kluyveri), a far relative of Saccharomyces cerevisiae, is not a widely studied organism in the laboratory. However, significant contributions to the understanding of nucleic acid precursors degradation in eukaryotes have been made using this model organism. Here we review eukaryotic pyrimidine degradation with emphasis on the contributions made with S. kluyveri and how this increases our understanding of human disease. Additionally, we discuss the possibilities and limitations of this nonconventional yeast as a laboratory organism.     

Amidohydrolases of the reductive pyrimidine catabolic pathway purification, characterization, structure, reaction mechanisms and enzyme deficiency

In the reductive pyrimidine catabolic pathway uracil and thymine are converted to beta-alanine and beta-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate (beta-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the beta-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. beta-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.     

Existence of cerebroside in Saccharomyces kluyveri and its related species

Sphingolipids are ubiquitous compounds derived from ceramide that consist of a sphingoid long-chain base with a 2-amino group amide linked to fatty acid and are present in the membranes of many organisms. As a principal sphingolipid, Saccharomyces cerevisiae contains a free ceramide and its inositol-phosphorylated derivatives (acidic types) but not a neutral glycosylated ceramide, glucosylceramide (cerebroside), which usually appears in eukaryotic cells. When 31 strains accepted in the genera Saccharomyces, Torulaspora, Zygosaccharomyces, and Kluyveromyces were analyzed for sphingolipids, cerebrosides were found in S. kluyveri, Z. cidri, Z. fermentati, K. lactis, K. thermotolerans, and K. waltii. The cerebrosides of S. kluyveri and K. lactis included 9-methyl 4-trans, 8-trans-sphingadienine and its putative metabolic intermediates. A unique characteristic of S. kluyveri was the presence of a trihydroxy sphingoid base, which rarely occurs in fungal cerebrosides. A polymerase chain reaction with primers targeted to the glucosylceramide synthase gene of other microorganisms amplified the fragments of the expected size from S. kluyveri and K. lactis and further extended to the adjacent regions. The presumed protein of S. kluyveri had 54.4% similarity to that of K. lactis, higher than the glucosylceramide synthases from Candida albicans, Pichia pastoris, and other organisms. From these observations, the divergence of S. kluyveri from the lineage of K. lactis in their evolution is discussed.     

Catabolism of pyrimidines in yeast: a tool to understand degradation of anticancer drugs

The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides/antibiotics. During the last decade we have developed a yeast species, Saccharomyces kluyveri, as a model and tool to study the genes and enzymes of the pyrimidine catabolic pathway. In this report, we studied degradation of uracil and its putative degradation products in 38 yeasts and showed that this pathway was present in the ancient yeasts but was lost approximately 100 million years ago in the S. cerevisiae lineage.     

A new expression vector for production of enzymes in the yeast Saccharomyces (Lachancea) kluyveri

We overexpressed and purified enzymes involved in the pyrimidine catabolic pathway in the yeast Saccharomyces (Lachancea) kluyveri. A new vector was therefore designed, providing the first specific expression system in Saccharomyces kluyveri. The URC1 gene was overexpressed and a soluble protein obtained and successfully purified using the C-terminally added His-tag. Our system will be used for further studies of the structure and function of the enzymes belonging to the URC pyrimidine degradation pathway.     

The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity

In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 angstroms, respectively. Both enzymes have a (beta/alpha)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller beta-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the beta-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-beta-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.     

Transformation of the yeast Saccharomyces kluyveri by Saccharomyces cerevisiae-based plasmids

For the transformation of the yeast Saccharomyces kluyveri, ura3 mutants were obtained by 5-fluoro-orotic acid selection. By utilizing the method based on treatment of intact cells with alkali cations, the ura3 strains of S. kluyveri were transformed by Saccharomyces cerevisiae-based plasmids. In the transformed cells, a S. cerevisiae centromere-based plasmid was stably replicated autonomously. Thus, this system will permit the study of gene expression and its regulation in S. kluyveri in relationship to that in S. cerevisiae.     

Yeast chromosomes have been significantly reshaped during their evolutionary history

The structure of the first eukaryotic genome, belonging to Saccharomyces cerevisiae, has been deduced; however, very little is known about its origin. In order to trace events that led to the current state of the Saccharomyces nuclear genomes, random fragments of genomic DNA from three yeasts were sequenced and compared to the S. cerevisiae database sequence. Whereas, S. cerevisiae and Saccharomyces bayanus show perfect synteny, a significant portion of the analysed fragments from Saccharomyces servazzii and Saccharomyces kluyveri show a different arrangement of genes when compared to S. cerevisiae. When the sequenced fragments were probed to the corresponding karyotype, a group of genes present on a single chromosome of S. servazzii and S. kluyveri had homologues scattered on several S. cerevisiae chromosomes. Apparently, extensive reorganisation of the chromosomes has taken place during evolution of the Saccharomyces yeasts. In addition, while one gross duplication could have taken place, at least a few genes have been duplicated independently at different time-points in the evolution.     

Degradation of pyrimidines in Saccharomyces kluyveri: transamination of beta-alanine

Beta-alanine is an intermediate in the reductive degradation of uracil. Recently we have identified and characterized the Saccharomyces kluyveri PYD4 gene and the corresponding enzyme beta -alanine aminotransferase ((Sk)Pyd4p), highly homologous to eukaryotic gamma-aminobutyrate aminotransferase (GABA-AT). S. kluyveri has two aminotransferases, GABA aminotransferase ((Sk)Uga1p) with 80% and (Sk)Pyd4p with 55% identity to S. cerevisiae GABA-AT. (Sk)Pyd4p is a typical pyridoxal phosphate-dependent aminotransferase, specific for alpha-ketoglutarate (alpha KG), beta-alanine (BAL) and gamma-aminobutyrate (GABA), showing a ping-pong kinetic mechanism involving two half-reactions and substrate inhibition. (Sk)Uga1p accepts only alpha KG and GABA but not BAL, thus only (Sk)Pydy4p belongs to the uracil degradative pathway.     

Production of fungal alpha-amylase by Saccharomyces kluyveri in glucose-limited cultivations

Heterologous protein production by the yeast Saccharomyces kluyveri was investigated under aerobic glucose-limited conditions. Alpha-amylase from Aspergillus oryzae was used as model protein and the gene was expressed from a S. cerevisiae 2 micro plasmid. For comparison, strains of both S. kluyveri and S. cerevisiae were transformed with the same plasmid, which led to secretion of active alpha-amylase in both cases. The S. cerevisiae 2 micro plasmid was found to be stable in S. kluyveri as evaluated by a constant alpha-amylase productivity in a continuous cultivation for more than 40 generations. S. kluyveri and S. cerevisiae secreted alpha-amylase with similar yields during continuous cultivations at dilution rates of 0.1 and 0.2 h(-1) (4.8-5.7 mg (g dry weight)(-1)). At a dilution rate of 0.3 h(-1) the metabolism of S. kluyveri was fully respiratory, whereas S. cerevisiae produced significant amounts of ethanol. A fed-batch cultivation was carried out with S. kluyveri where the biomass concentration reached 85 g l(-1) and the alpha-amylase concentration reached 320 mg l(-1). Even though S. kluyveri could be grown to high cell density, it was also observed that it has a high maintenance coefficient, which resulted in low biomass yields at the low specific growth rates prevailing towards the end of the fed-batch cultivation.     

Peptide inhibitors of appressorium development in Glomerella cingulata

The phytopathogen Glomerella cingulata (anamorph: Colletotrichum gloeosporioides) infects host tissue by means of a specialised infection structure, the appressorium. The Saccharomyces cerevisiae alpha-mating factor pheromone, the Saccharomyces kluyveri alpha-mating factor pheromone and a hendecapeptide produced by G. cingulata inhibit appressorium development. The amino acid sequence of the G. cingulata peptide, GYFSYPHGNLF, is different from that of the mature pheromone peptides of other filamentous fungi. The peptide has sequence similarity with the Saccharomyces alpha-mating factor pheromones, but is unable to elicit growth arrest in S. cerevisiae.     

Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts

Translation of mitochondrially coded mRNAs in Saccharomyces cerevisiae depends on membrane-bound mRNA-specific activator proteins, whose targets lie in the mRNA 5'-untranslated leaders (5'-UTLs). In at least some cases, the activators function to localize translation of hydrophobic proteins on the inner membrane and are rate limiting for gene expression. We searched unsuccessfully in divergent budding yeasts for orthologs of the COX2- and COX3-specific translational activator genes, PET111, PET54, PET122, and PET494, by direct complementation. However, by screening for complementation of mutations in genes adjacent to the PET genes in S. cerevisiae, we obtained chromosomal segments containing highly diverged homologs of PET111 and PET122 from Saccharomyces kluyveri and of PET111 from Kluyveromyces lactis. All three of these genes failed to function in S. cerevisiae. We also found that the 5'-UTLs of the COX2 and COX3 mRNAs of S. kluyveri and K. lactis have little similarity to each other or to those of S. cerevisiae. To determine whether the PET111 and PET122 homologs carry out orthologous functions, we deleted them from the S. kluyveri genome and deleted PET111 from the K. lactis genome. The pet111 mutations in both species prevented COX2 translation, and the S. kluyveri pet122 mutation prevented COX3 translation. Thus, while the sequences of these translational activator proteins and their 5'-UTL targets are highly diverged, their mRNA-specific functions are orthologous.     

Ability for anaerobic growth is not sufficient for development of the petite phenotype in Saccharomyces kluyveri

Saccharomyces cerevisiae is a petite-phenotype-positive ("petite-positive") yeast, which can successfully grow in the absence of oxygen. On the other hand, Kluyveromyces lactis as well as many other yeasts are petite negative and cannot grow anaerobically. In this paper, we show that Saccharomyces kluyveri can grow under anaerobic conditions, but while it can generate respiration-deficient mutants, it cannot generate true petite mutants. From a phylogenetic point of view, S. kluyveri is apparently more closely related to S. cerevisiae than to K. lactis. These observations suggest that the progenitor of the modern Saccharomyces and Kluyveromyces yeasts, as well as other related genera, was a petite-negative and aerobic yeast. Upon separation of the K. lactis and S. kluyveri-S. cerevisiae lineages, the latter developed the ability to grow anaerobically. However, while the S. kluyveri lineage has remained petite negative, the lineage leading to the modern Saccharomyces sensu stricto and sensu lato yeasts has developed the petite-positive characteristic.     

Production of a heterologous proteinase A by Saccharomyces kluyveri

In order to evaluate the potential of Saccharomyces kluyveri for heterologous protein production, S. kluyveri Y159 was transformed with a S. cerevisiae-based multi-copy plasmid containing the S. cerevisiae PEP4 gene, which encodes proteinase A, under the control of its native promoter. As a reference, S. cerevisiae CEN.PK 113-5D was transformed with the same plasmid and the two strains were characterised in batch cultivations on glucose. The glucose metabolism was found to be less fermentative in S. kluyveri than in S. cerevisiae. The yield of ethanol on glucose was 0.11 g/g in S. kluyveri, compared to a yield of 0.40 g/g in S. cerevisiae. Overexpression of PEP4 led to the secretion of active proteinase A in both S. kluyveri and S. cerevisiae. The yield of active proteinase A during growth on glucose was found to be 3.6-fold higher in S. kluyveri than in the S. cerevisiae reference strain.