| 1. |
Lundgren S,
Gojkovi? Z,
Piskur J,
Dobritzsch D,
( 2003 ) Yeast beta-alanine synthase shares a structural scaffold and origin with dizinc-dependent exopeptidases. PMID : 14534321 : DOI : 10.1074/jbc.M308674200 Abstract >>
beta-Alanine synthase (beta AS) is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of pyrimidine bases, including several anticancer drugs. In eukaryotes, beta ASs belong to two subfamilies, which exhibit a low degree of sequence similarity. We determined the structure of beta AS from Saccharomyces kluyveri to a resolution of 2.7 A. The subunit of the homodimeric enzyme consists of two domains: a larger catalytic domain with a dizinc metal center, which represents the active site of beta AS, and a smaller domain mediating the majority of the intersubunit contacts. Both domains exhibit a mixed alpha/beta-topology. Surprisingly, the observed high structural homology to a family of dizinc-dependent exopeptidases suggests that these two enzyme groups have a common origin. Alterations in the ligand composition of the metal-binding site can be explained as adjustments to the catalysis of a different reaction, the hydrolysis of an N-carbamyl bond by beta AS compared with the hydrolysis of a peptide bond by exopeptidases. In contrast, there is no resemblance to the three-dimensional structure of the functionally closely related N-carbamyl-d-amino acid amidohydrolases. Based on comparative structural analysis and observed deviations in the backbone conformations of the eight copies of the subunit in the asymmetric unit, we suggest that conformational changes occur during each catalytic cycle.
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2. |
Kurtzman CP,
Robnett CJ,
( 2003 ) Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses. PMID : 12748053 : DOI : 10.1016/S1567-1356(03)00012-6 Abstract >>
Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.
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3. |
Takakuwa N,
Kinoshita M,
Oda Y,
Ohnishi M,
( 2002 ) Existence of cerebroside in Saccharomyces kluyveri and its related species. PMID : 12702269 : DOI : 10.1111/j.1567-1364.2002.tb00120.x Abstract >>
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.
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4. |
Langkjaer RB,
Cliften PF,
Johnston M,
Piskur J,
( 2003 ) Yeast genome duplication was followed by asynchronous differentiation of duplicated genes. PMID : 12594514 : DOI : 10.1038/nature01419 Abstract >>
Gene redundancy has been observed in yeast, plant and human genomes, and is thought to be a consequence of whole-genome duplications. Baker's yeast, Saccharomyces cerevisiae, contains several hundred duplicated genes. Duplication(s) could have occurred before or after a given speciation. To understand the evolution of the yeast genome, we analysed orthologues of some of these genes in several related yeast species. On the basis of the inferred phylogeny of each set of genes, we were able to deduce whether the gene duplicated and/or specialized before or after the divergence of two yeast lineages. Here we show that the gene duplications might have occurred as a single event, and that it probably took place before the Saccharomyces and Kluyveromyces lineages diverged from each other. Further evolution of each duplicated gene pair-such as specialization or differentiation of the two copies, or deletion of a single copy--has taken place independently throughout the evolution of these species.
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5. |
Daniel HM,
Sorrell TC,
Meyer W,
( 2001 ) Partial sequence analysis of the actin gene and its potential for studying the phylogeny of Candida species and their teleomorphs. PMID : 11491363 : DOI : 10.1099/00207713-51-4-1593 Abstract >>
The actin gene has been studied as a potential phylogenetic marker for selected members of the anamorphic genus Candida and seven related teleomorphic genera (Debaryomyces, Issatchenkia, Kluyveromyces, Saccharomyces and Pichia from the Saccharomycetaceae; Clavispora and Metschnikowia from the Metschnikowiaceae). The nucleotide sequences of 36 fungal taxa were analysed with respect to their molecular evolution and phylogenetic relationships. A total of 460 bp (47%) of the coding 979 bp were variable and 396 bp (40%) of these were found to be phylogenetically informative. Further analysis of the sequences showed that the genic G+C contents were higher than the nuclear G+C contents for most of the taxa. A strong positive correlation was found between G+C content over all codon positions and third positions. First and second codon positions were considered to be independent of the genic G+C content. The expected transition/transversion bias was detected only for third positions. Pairwise comparisons of transitional and transversional changes (substitutions) with total percentage sequence divergences revealed that the third position transitions showed no saturation for ingroup comparisons. A specific weighting scheme was set up, combining codon-position weights with change-frequency weights to enable the inclusion of distant outgroup taxa. Parsimony analyses of the investigated taxa showed four groups, three of which corresponded to major clusters that had been established previously in Candida by rDNA analysis. Interrelationships among the species groups in this heterogeneous anamorphic genus were determined. The polyphyletic origin of the selected Candida species and their close associations with several ascomycete genera were verified and known anamorph/teleomorph pairs confirmed. The actin gene was established as a valuable phylogenetic marker with the particular advantage of an unambiguous alignment.
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6. |
Gojkovi? Z,
Sandrini MP,
Piskur J,
( 2001 ) Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity. PMID : 11454750 : PMC : PMC1461717 Abstract >>
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.
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7. |
Oda Y,
Fujisawa T,
( 2001 ) Intraspecific divergence of Saccharomyces kluyveri as revealed by the nucleotide sequences of 18S-28S rRNA spacer regions and alpha-galactosidase MEL genes. PMID : 11272822 : Abstract >>
In the five strains classified as the yeast Saccharomyces kluyveri, several substitutions were observed in the two internal transcribed spacer regions between 18S and 28S rRNA. A PCR reaction with primers targeted to the MEL1 gene of Saccharomyces cerevisiae amplified fragments of the expected size, and those sequences showed significant divergence in the strains of S. kluyveri.
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8. |
Querol A,
Belloch C,
( 2000 ) Phylogeny of the genus Kluyveromyces inferred from the mitochondrial cytochrome-c oxidase II gene. PMID : 10826829 : DOI : 10.1099/00207713-50-1-405 Abstract >>
A phylogenetic analysis of 17 species belonging to the genus Kluyveromyces and 12 reference and outgroup species was performed using mitochondrial cytochrome-c oxidase II gene sequences. The genus Kluyveromyces appears as a polyphyletic taxon formed by species included within the following four main groups. The Kluyveromyces phaffii group encompasses the species Kluyveromyces blattae, K. phaffii and Kluyveromyces yarrowii. The Kluyveromyces marxianus group is a monophyletic group consisting of the species Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, K. marxianus and Kluyveromyces wickerhamii. The monophyletic Kluyveromyces thermotolerans group is formed by K. thermotolerans, Kluyveromyces waltii and Saccharomyces kluyveri (which appears in the mitochondrial tree as the sister clade of the K. marxianus group). Finally, the Saccharomyces cerevisiae group contains the remaining Kluyveromyces species, as well as the reference Saccharomyces species (sensu lato and sensu stricto) and Candida glabrata (the phylogenetic relationships within this group are unclear according to the bootstrap test). The phylogenetic relationships obtained for this mitochondrial gene are, for the most part, congruent with previous trees based on nuclear rRNA sequences, except for the position of K. yarrowii and the close relationship between the K. marxianus and K. thermotolerans groups. These differences, as well as the existence of these groups, are discussed in the context of previous studies based on phenotypic, genetic and molecular data. Although additional studies are required to decipher the phylogenetic relationships between the genus Kluyveromyces and the closely related genera Saccharomyces, Torulaspora and Zygosaccharomyces, future changes to their taxonomic status should take account of the existence of these four groups of Kluyveromyces species.
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9. |
Sloan J,
Miller B,
Young ET,
( 2000 ) Evolution of a glucose-regulated ADH gene in the genus Saccharomyces. PMID : 10717481 : DOI : 10.1016/s0378-1119(00)00035-4 Abstract >>
To determine when a glucose-repressed alcohol dehydrogenase isozyme and its regulatory gene, ADR1, arose during evolution, we surveyed species of the genus Saccharomyces for glucose-repressed ADH isozymes and for ADR1 homologues. Glucose-repressed ADH isozymes were present in all species of Saccharomyces sensu strictu and also in Saccharomyces kluyveri, the most distant member of the Saccharomyces clade. We cloned and characterized ADH promoters from S. bayanus, S. douglasii, and S. kluyveri. The ADH promoters from S. bayanus and S. douglasii had conserved sequences, including upstream regulatory elements, and an extended polydA tract. The expression of a reporter gene driven by the S. bayanus promoter was glucose-repressed and dependent on the major activator of transcription, ADR1, when it was introduced into S. cerevisiae. One S. kluyveri promoter was also glucose-repressed and ADR1-dependent in S. cerevisiae. The other S. kluyveri ADH promoter was expressed constitutively and was ADR1-independent. Although showing little sequence conservation with the S. cerevisiae ADH2 promoter, the glucose-repressed S. kluyveri promoter contains numerous potential binding sites for Adr1. The glucose-repressed ADH from S. kluyveri is a mitochondrial isozyme most closely related to S. cerevisiae ADHIII. ADR1 homologues from S. douglasii and S. paradoxus contain a trinucleotide repeat encoding polyAsn that is lacking in S. cerevisiae and S. bayanus. No ADR1 homologue could be detected in S. kluyveri, suggesting that the potential for Adr1 regulation may have arisen first, before ADR1 evolved.
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10. |
Bonnefoy N,
Williams EH,
Costanzo MC,
( 2000 ) Highly diverged homologs of Saccharomyces cerevisiae mitochondrial mRNA-specific translational activators have orthologous functions in other budding yeasts. PMID : 10757749 : PMC : PMC1460983 Abstract >>
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.
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11. |
Andersen G,
Andersen B,
Dobritzsch D,
Schnackerz KD,
Piskur J,
( 2007 ) A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast. PMID : 17355287 : DOI : 10.1111/j.1742-4658.2007.05729.x Abstract >>
In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.
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12. |
Oura T,
Kajiwara S,
( 2006 ) Cloning and functional characterization of a fatty acid synthase component FAS2 gene from Saccharomyces kluyveri. PMID : 16479401 : DOI : 10.1007/s00294-006-0063-4 Abstract >>
A gene coding the alpha subunit of fatty acid synthase (FAS2) was isolated from the budding yeast Saccharomyces kluyveri. Nucleotide sequence analysis indicated that this gene, termed Sk-FAS2, coded a protein having an amino acid sequence 83% identical to the FAS2 protein of S. cerevisiae (Sc-FAS2). The Sk-FAS2 gene was able to functionally complement an S. cerevisiae fas2 disruptant. This Sk-FAS2-expressing strain was found to produce larger amounts of C18 than C16, in contrast to the Sc-FAS2-expressing fas2 strain. In addition, fusion genes of Sk-FAS2 and Sc-FAS2 were transformed into a fas2-disrupted strain of S. cerevisiae, and fatty acid analysis of these transformants suggested that the region containing the acyl carrier protein and beta-ketoacyl reductase domains of yeast FAS2 protein play an important role in determining carbon chain length of fatty acids.
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13. |
Prigozy T,
Gonzales E,
Broek D,
( 1992 ) Identification and analysis of a DNA fragment from Saccharomyces kluyveri that can complement the loss of CDC25 function in Saccharomyces cerevisiae. PMID : 1644315 : DOI : 10.1016/0378-1119(92)90491-7 Abstract >>
In the budding yeast, Saccharomyces cerevisiae, the function of wild-type Ras proteins is dependent on the CDC25 protein, which promotes the exchange of guanine nucleotides bound to Ras. To facilitate the identification of proteins which similarly regulate Ras function in higher eukaryotes, we have identified the CDC25 gene from another budding yeast, Saccharomyces kluyveri, by low-stringency hybridization to an S. cerevisiae CDC25 restriction fragment. This protein, SKCDC25, shares significant amino acid homology with CDC25, SCD25, and Ste6 of Schizosaccharomyces pombe in the C-terminal portion of the protein. The expression of SKCDC25 in a temperature-sensitive cdc25 strain of S. cerevisiae complements the loss of endogenous CDC25 activity. The identification of the highly conserved C-terminal sequences, which direct bona fide CDC25 activity within these proteins, will aid in the isolation of CDC25 genes from higher eukaryotes.
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14. |
Thomson JM,
Gaucher EA,
Burgan MF,
De Kee DW,
Li T,
Aris JP,
Benner SA,
( 2005 ) Resurrecting ancestral alcohol dehydrogenases from yeast. PMID : 15864308 : DOI : 10.1038/ng1553 PMC : PMC3618678 Abstract >>
Modern yeast living in fleshy fruits rapidly convert sugars into bulk ethanol through pyruvate. Pyruvate loses carbon dioxide to produce acetaldehyde, which is reduced by alcohol dehydrogenase 1 (Adh1) to ethanol, which accumulates. Yeast later consumes the accumulated ethanol, exploiting Adh2, an Adh1 homolog differing by 24 (of 348) amino acids. As many microorganisms cannot grow in ethanol, accumulated ethanol may help yeast defend resources in the fruit. We report here the resurrection of the last common ancestor of Adh1 and Adh2, called Adh(A). The kinetic behavior of Adh(A) suggests that the ancestor was optimized to make (not consume) ethanol. This is consistent with the hypothesis that before the Adh1-Adh2 duplication, yeast did not accumulate ethanol for later consumption but rather used Adh(A) to recycle NADH generated in the glycolytic pathway. Silent nucleotide dating suggests that the Adh1-Adh2 duplication occurred near the time of duplication of several other proteins involved in the accumulation of ethanol, possibly in the Cretaceous age when fleshy fruits arose. These results help to connect the chemical behavior of these enzymes through systems analysis to a time of global ecosystem change, a small but useful step towards a planetary systems biology.
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15. |
Diezmann S,
Cox CJ,
Schönian G,
Vilgalys RJ,
Mitchell TG,
( 2004 ) Phylogeny and evolution of medical species of Candida and related taxa: a multigenic analysis. PMID : 15583292 : DOI : 10.1128/JCM.42.12.5624-5635.2004 PMC : PMC535224 Abstract >>
Hemiascomycetes are species of yeasts within the order Saccharomycetales. The order encompasses disparate genera with a variety of life styles, including opportunistic human pathogens (e.g., Candida albicans), plant pathogens (e.g., Eremothecium gossypii), and cosmopolitan yeasts associated with water and decaying vegetation. To analyze the phylogeny of medically important species of yeasts, we selected 38 human pathogenic and related strains in the order Saccharomycetales. The DNA sequences of six nuclear genes were analyzed by maximum likelihood and Bayesian phylogenetic methods. The maximum likelihood analysis of the combined data for all six genes resolved three major lineages with significant support according to Bayesian posterior probability. One clade was mostly comprised of pathogenic species of Candida. Another major group contained members of the family Metschnikowiaceae as a monophyletic group, three species of Debaryomyces, and strains of Candida guilliermondii. The third clade consisted exclusively of species of the family Saccharomycetaceae. Analysis of the evolution of key characters indicated that both codon reassignment and coenzyme Q(9) likely had single origins with multiple losses. Tests of correlated character evolution revealed that these two traits evolved independently.
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16. |
Watanabe K,
Oura T,
Sakai H,
Kajiwara S,
( 2004 ) Yeast Delta 12 fatty acid desaturase: gene cloning, expression, and function. PMID : 15056908 : DOI : 10.1271/bbb.68.721 Abstract >>
In an effort to elucidate the molecular mechanisms of fatty acid desaturation in yeast, a complete gene encoding Delta 12 fatty acid desaturase of Saccharomyces kluyveri was cloned. The open reading frame of this gene (named Sk-FAD2) consists of 1,251 bp, encoding 416 amino acids. The deduced Sk-FAD2 protein had 37-55% identity with those from other filamentous fungi. Unlike the genes of these other fungi, S. cerevisiae expressing Sk-FAD2 was found to be capable of synthesizing the dienoic fatty acid hexadecadienoic acid as well as linoleic acid. Moreover, the Sk-FAD2-disrupted strain of S. kluyveri was unable to produce polyunsaturated fatty acids. These results suggested that Sk-FAD2 protein is a unique Delta 12 fatty acid desaturase in S. kluyveri. Analysis of transcriptional expression revealed that Sk-FAD2 was not repressed by exogenous unsaturated fatty acids but responded to low-temperature stress.
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17. |
Møller K,
Langkjaer RB,
Nielsen J,
Piskur J,
Olsson L,
( 2004 ) Pyruvate decarboxylases from the petite-negative yeast Saccharomyces kluyveri. PMID : 14648197 : DOI : 10.1007/s00438-003-0950-z Abstract >>
Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S. kluyveri and S. cerevisiae with respect to ethanol formation under aerobic conditions could be caused by differences in the regulation of this enzyme activity. We have identified and cloned three genes encoding functional pyruvate decarboxylase enzymes (PDCgenes) from the type strain of S. kluyveri (Sk- PDC11, Sk- PDC12 and Sk- PDC13). The regulation of pyruvate decarboxylase in S. kluyveri was studied by measuring the total level of Sk- PDC mRNA and the overall enzyme activity under various growth conditions. It was found that the level of Sk- PDC mRNA was enhanced by glucose and oxygen limitation, and that the level of enzyme activity was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/ Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae and S. kluyveri each have three PDC genes, these have apparently arisen by independent duplications and specializations in each of the two yeast lineages.
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18. |
Young D,
O'Neill K,
Broek D,
Wigler M,
( 1991 ) The adenylyl cyclase-encoding gene from Saccharomyces kluyveri. PMID : 1864503 : DOI : 10.1016/0378-1119(91)90551-l Abstract >>
The gene encoding adenylyl cyclase (CYR) from Saccharomyces kluyveri has been cloned. Comparison of the predicted amino acid sequence of this protein with the Schizosaccharomyces pombe and Saccharomyces cerevisiae CYRs revealed homology between different structural and putative functional domains that suggest a high degree of conservation in the function and regulation of these proteins.
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19. |
Andersen G,
Björnberg O,
Polakova S,
Pynyaha Y,
Rasmussen A,
Møller K,
Hofer A,
Moritz T,
Sandrini MP,
Merico AM,
Compagno C,
Akerlund HE,
Gojkovi? Z,
Piskur J,
( 2008 ) A second pathway to degrade pyrimidine nucleic acid precursors in eukaryotes. PMID : 18550080 : DOI : 10.1016/j.jmb.2008.05.029 Abstract >>
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.
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20. |
Andersen B,
Lundgren S,
Dobritzsch D,
Piskur J,
( 2008 ) A recruited protease is involved in catabolism of pyrimidines. PMID : 18448119 : DOI : 10.1016/j.jmb.2008.03.073 Abstract >>
In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.
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21. |
Lundgren S,
Andersen B,
Piskur J,
Dobritzsch D,
( 2007 ) Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements. PMID : 17916556 : DOI : 10.1074/jbc.M705517200 Abstract >>
Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.
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22. |
Marsh L,
Herskowitz I,
( 1988 ) STE2 protein of Saccharomyces kluyveri is a member of the rhodopsin/beta-adrenergic receptor family and is responsible for recognition of the peptide ligand alpha factor. PMID : 2836861 : DOI : 10.1073/pnas.85.11.3855 PMC : PMC280318 Abstract >>
We have cloned the gene for the alpha-factor receptor of the yeast Saccharomyces kluyveri by using the Saccharomyces cerevisiae receptor gene (c-STE2) as a probe. The nucleotide sequence of the S. kluyveri gene (k-STE2) shows that its predicted polypeptide contains seven hydrophobic segments capable of spanning a lipid bilayer and thus that, like c-STE2, it appears to be a member of the rhodopsin/beta-adrenergic receptor family. The k-STE2 polypeptide is 50% identical to that coded by c-STE2, with high conservation (greater than 67%) in the putative membrane-spanning domains. The carboxyl-terminal amino acid sequences are not similar, but both are very hydrophilic and rich in serine and threonine residues. The k-STE2 gene is functional in S. cerevisiae: it reverses the mating defect of an S. cerevisiae mutant defective in its STE2 gene. S. cerevisiae strains expressing k-STE2 rather than c-STE2 exhibit the mating-factor selectivity characteristic of S. kluyveri: better response to S. kluyveri alpha factor than to S. cerevisiae alpha factor. (S. cerevisiae normally responds much better to its own alpha-factor peptide than to the related alpha-factor peptide of S. kluyveri.) This observation demonstrates that the STE2 gene is responsible for ligand selectivity and provides additional evidence that the STE2 protein is the receptor for alpha factor.
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23. |
Melnikov S,
Mailliot J,
Rigger L,
Neuner S,
Shin BS,
Yusupova G,
Dever TE,
Micura R,
Yusupov M,
( 2016 ) Molecular insights into protein synthesis with proline residues. PMID : 27827794 : DOI : 10.15252/embr.201642943 PMC : PMC5283605 Abstract >>
Proline is an amino acid with a unique cyclic structure that facilitates the folding of many proteins, but also impedes the rate of peptide bond formation by the ribosome. As a ribosome substrate, proline reacts markedly slower when compared with other amino acids both as a donor and as an acceptor of the nascent peptide. Furthermore, synthesis of peptides with consecutive proline residues triggers ribosome stalling. Here, we report crystal structures of the eukaryotic ribosome bound to analogs of mono- and diprolyl-tRNAs. These structures provide a high-resolution insight into unique properties of proline as a ribosome substrate. They show that the cyclic structure of proline residue prevents proline positioning in the amino acid binding pocket and affects the nascent peptide chain position in the ribosomal peptide exit tunnel. These observations extend current knowledge of the protein synthesis mechanism. They also revise an old dogma that amino acids bind the ribosomal active site in a uniform way by showing that proline has a binding mode distinct from other amino acids.
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24. |
Aylett CH,
Boehringer D,
Erzberger JP,
Schaefer T,
Ban N,
( 2015 ) Structure of a yeast 40S-eIF1-eIF1A-eIF3-eIF3j initiation complex. PMID : 25664723 : DOI : 10.1038/nsmb.2963 Abstract >>
Eukaryotic translation initiation requires cooperative assembly of a large protein complex at the 40S ribosomal subunit. We have resolved a budding yeast initiation complex by cryo-EM, allowing placement of prior structures of eIF1, eIF1A, eIF3a, eIF3b and eIF3c. Our structure highlights differences in initiation-complex binding to the ribosome compared to that of mammalian eIF3, demonstrates a direct contact between eIF3j and eIF1A and reveals the network of interactions between eIF3 subunits.
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25. |
( 1996 ) Functional conservation of yeast mtTFB despite extensive sequence divergence. PMID : 9196077 : PMC : PMC6148273 Abstract >>
Transcription of mtDNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mtRNA polymerase acting with a 40-kDa dissociable factor known as mtTFB or Mtflp. mtTFB has been cloned and characterized in S. cerevisiae, but has not been studied in similar detail in any other organism. Although it is known that mitochondrial transcription in the dairy yeast, Kluyveromyces lactis, initiates within the same consensus promoter sequence used in S. cerevisiae, no previous studies have focused on the proteins involved in transcription initiation in K. lactis. In this article, we report the cloning of mtTFB from K. lactis and from a yeast more closely related to S. cerevisiae, S. kluyveri. Both novel mtTFB genes were able to substitute for the MTF1 gene in S. cerevisiae. Both proteins purified following expression in E. coli were able to support specific transcription initiation in vitro with the S. cerevisiae mtRNA polymerase. The S. kluyveri and K. lactis mtTFB proteins share only 56% and 40% identity with S. cerevisiae mtTFB, respectively. Alignments of the three mtTFB sequences did not reveal any regions larger than 30 amino acids with greater than 60% amino acid identity. In particular, regions proposed to show sequence similarity to bacterial sigma factors were not more highly conserved than other regions of the mtTFB proteins. All three yeast mtTFB genes lack conventional amino-terminal mitochondrial targeting sequences, suggesting that all three proteins may be imported into mitochondria by the same unusual mechanism reported for S. cerevisiae mtTFB.
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26. |
Egel-Mitani M,
Hansen MT,
( 1987 ) Nucleotide sequence of the gene encoding the Saccharomyces kluyveri alpha mating pheromone. PMID : 3627991 : DOI : 10.1093/nar/15.15.6303 PMC : PMC306093 Abstract >>
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