Functional studies of aldo-keto reductases in Saccharomyces cerevisiae

We utilized the budding yeast Saccharomyces cerevisiae as a model to systematically explore physiological roles for yeast and mammalian aldo-keto reductases. Six open reading frames encoding putative aldo-keto reductases were identified when the yeast genome was queried against the sequence for human aldose reductase, the prototypical mammalian aldo-keto reductase. Recombinant proteins produced from five of these yeast open reading frames demonstrated NADPH-dependent reductase activity with a variety of aldehyde and ketone substrates. A triple aldo-keto reductase null mutant strain demonstrated a glucose-dependent heat shock phenotype which could be rescued by ectopic expression of human aldose reductase. Catalytically-inactive mutants of human or yeast aldo-keto reductases failed to effect a rescue of the heat shock phenotype, suggesting that the phenotype results from either an accumulation of one or more unmetabolized aldo-keto reductase substrates or a synthetic deficiency of aldo-keto reductase products generated in response to heat shock stress. These results suggest that multiple aldo-keto reductases fulfill functionally redundant roles in the stress response in yeast.     

Microbial aldo-keto reductases

The aldo-keto reductases (AKR) are a superfamily of enzymes with diverse functions in the reduction of aldehydes and ketones. AKR enzymes are found in a wide range of microorganisms, and many open reading frames encoding related putative enzymes have been identified through genome sequencing projects. Established microbial members of the superfamily include the xylose reductases, 2,5-diketo-D-gluconic acid reductases and beta-keto ester reductases. The AKR enzymes share a common (alpha/beta)(8) structure, and conserved catalytic mechanism, although there is considerable variation in the substrate-binding pocket. The physiological function of many of these enzymes is unknown, but a variety of methods including gene disruptions, heterologous expression systems and expression profiling are being employed to deduce the roles of these enzymes in cell metabolism. Several microbial AKR are already being exploited in biotransformation reactions and there is potential for other novel members of this important superfamily to be identified, studied and utilized in this way.     

Exopolyphosphatases of the yeast Saccharomyces cerevisiae

Separate compartments of the yeast cell possess their own exopolyphosphatases differing from each other in their properties and dependence on culture conditions. The low-molecular-mass exopolyphosphatases of the cytosol, cell envelope, and mitochondrial matrix are encoded by the PPX1 gene, while the high-molecular-mass exopolyphosphatase of the cytosol and those of the vacuoles, mitochondrial membranes, and nuclei are presumably encoded by their own genes. Based on recent works, a preliminary classification of the yeast exopolyphosphatases is proposed.     

Functional genomic studies of aldo-keto reductases

Aldose reductase (AR) is considered a potential mediator of diabetic complications and is a drug target for inhibitors of diabetic retinopathy and neuropathy in clinical trials. However, the physiological role of this enzyme still has not been established. Since effective inhibition of diabetic complications will require early intervention, it is important to delineate whether AR fulfills a physiological role that cannot be compensated by an alternate aldo-keto reductase. Functional genomics provides a variety of powerful new tools to probe the physiological roles of individual genes, especially those comprising gene families. Several eucaryotic genomes have been sequenced and annotated, including yeast, nematode and fly. To probe the function of AR, we have chosen to utilize the budding yeast Saccharomyces cerevisiae as a potential model system. Unlike Caenorhabditis elegans and D. melanogaster, yeast provides a more desirable system for our studies because its genome is manipulated more readily and is able to sustain multiple gene deletions in the presence of either drug or auxotrophic selectable markers. Using BLAST searches against the human AR gene sequence, we identified six genes in the complete S. cerevisiae genome with strong homology to AR. In all cases, amino acids thought to play important catalytic roles in human AR are conserved in the yeast AR-like genes. All six yeast AR-like open reading frames (ORFs) have been cloned into plasmid expression vectors. Substrate and AR inhibitor specificities have been surveyed on four of the enzyme forms to identify, which are the most functionally similar to human AR. Our data reveal that two of the enzymes (YDR368Wp and YHR104Wp) are notable for their similarity to human AR in terms of activity with aldoses and substituted aromatic aldehydes. Ongoing studies are aimed at characterizing the phenotypes of yeast strains containing single and multiple knockouts of the AR-like genes.     

Iron-reductases in the yeast Saccharomyces cerevisiae

Several NAD(P)H-dependent ferri-reductase activities were detected in sub-cellular extracts of the yeast Saccharomyces cerevisiae. Some were induced in cells grown under iron-deficient conditions. At least two cytosolic iron-reducing enzymes having different substrate specificities could contribute to iron assimilation in vivo. One enzyme was purified to homogeneity: it is a flavoprotein (FAD) of 40 kDa that uses NADPH as electron donor and Fe(III)-EDTA as artificial electron acceptor. Isolated mitochondria reduced a variety of ferric chelates, probably via an 'external' NADH dehydrogenase, but not the siderophore ferrioxamine B. A plasma membrane-bound ferri-reductase system functioning with NADPH as electron donor and FMN as prosthetic group was purified 100-fold from isolated plasma membranes. This system may be involved in the reductive uptake of iron in vivo.     

Meiotic karyotype of the yeast Saccharomyces cerevisiae

A cytogenetic study of the meiotic chromosomes of the budding yeast Saccharomyces cerevisiae was undertaken by high resolution epifluorescence microscopy. Condensation of chromatin into separate chromosomes takes place during prophase I. At metaphase I, there are 16 separate and distinct bivalents which are roughly classified into three groups by morphological differences and DNA content.     

Damage-induced recombination in the yeast Saccharomyces cerevisiae

Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity following DNA damage from endogenous and exogenous genotoxic sources. One of the mechanisms used to repair damaged chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a damaged region of the genome. In this review, I summarized what is known about the molecular and cellular mechanisms by which various DNA-damaging agents induce recombination in yeast. The yeast Saccharomyces cerevisiae has served as an excellent model organism to study the induction of recombination. It has helped to define the basic phenomenology and to isolate the genes involved in the process. Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans. Many carcinogens are known to induce recombination and to cause chromosomal rearrangements. An understanding of the mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans.     

Sporulation in the budding yeast Saccharomyces cerevisiae

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.     

Engineering steroid hormone specificity into aldo-keto reductases

Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs), 20alpha-HSDs, as well as the 5beta-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3alpha-HSD (AKR1C9) as a starting structure to engineer either 5beta-reductase or 20alpha-HSD activity. 5beta-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a k(cat) comparable to that for homogeneous rat and human liver 5beta-reductases. pH versus k(cat) profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH(2)(+) character facilitates enolization of the Delta(4)-3-ketosteroid and subsequent hydride transfer to C5. Since 5beta-reductase precedes 3alpha-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3alpha-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20alpha-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log(10)(k(cat)/K(m)) from androgens to progestins was of the order of 10(11). This represents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs will be ultimately rationalized.     

Engineering steroid hormone specificity into aldo-keto reductases

Steroid hormone transforming aldo-keto reductases (AKRs) include virtually all mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs), 20α-HSDs, as well as the 5β-reductases. To elucidate the molecular determinants of steroid hormone recognition we used rat liver 3α-HSD (AKR1C9) as a starting structure to engineer either 5β-reductase or 20α-HSD activity. 5β-Reductase activity was introduced by a single point mutation in which the conserved catalytic His (H117) was mutated to Glu117. The H117E mutant had a k_cat comparable to that for homogeneous rat and human liver 5β-reductases. pH versus k_cat profiles show that this mutation increases the acidity of the catalytic general acid Tyr55. It is proposed that the increased TyrOH₂⁺ character facilitates enolization of the Δ⁴-3-ketosteroid and subsequent hydride transfer to C5. Since 5β-reductase precedes 3α-HSD in steroid hormone metabolism it is likely that this metabolic pathway arose by gene duplication and point mutation. 3α-HSD is positional and stereospecific for 3-ketosteroids and inactivates androgens. The enzyme was converted to a robust 20α-HSD, which is positional and stereospecific for 20-ketosteroids and inactivates progesterone, by the generation of loop-chimeras. The shift in log₁₀(k_cat/K_m) from androgens to progestins was of the order of 10¹¹. This represents a rare example of how steroid hormone specificity can be changed at the enzyme level. Protein engineering with predicted outcomes demonstrates that the molecular determinants of steroid hormone recognition in AKRs will be ultimately rationalized.     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.     

Aquaporins in Saccharomyces cerevisiae wine yeast

AQY1 and AQY2 were sequenced from five commercial and five native wine yeasts. Of these, two AQY1 alleles from UCD 522 and UCD 932 were identified that encoded three or four amino-acid changes, respectively, compared with the Sigma1278b sequence. Oocytes expressing these AQY1 alleles individually exhibited increased water permeability vs. water-injected oocytes, whereas oocytes expressing the AQY2 allele from UCD 932 did not show an increase, as expected, owing to an 11 bp deletion. Wine strains lacking Aqy1p did not show a decrease in spore fitness or enological aptitude under stressful conditions, limited nitrogen, or increased temperature. The exact role of aquaporins in wine yeasts remains unclear.     

Immunochemical characterization of aldo-keto reductases from human tissues

Aldose reductase, aldehyde reductase and carbonyl reductase constitute a family of monomeric NADPH-dependent oxidoreductases with similar physical and chemical properties. Characterization of the enzymes from human tissues by immunotitration and an enzyme immunoassay indicated that, despite their apparent likeness, the three reductases do not cross-react immunochemically.     

Atg9 trafficking in the yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and nonselective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element. Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.     

Advanced biofuel production by the yeast Saccharomyces cerevisiae

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Acid excreting mutants of yeast Saccharomyces cerevisiae

Saccharomyces cerevisiae mutants acidifying glucose medium containing bromocresol purple were shown to excrete protons when placed in unbuffered water in the absence of any external carbon source. The mutants belong to 16 different complementation groups. Most of them do not grow on glycerol and the excreted protons are associated to particular sets of organic anions such as citrate, aconitate, succinate, fumarate or malate. These novel types of respiratory mutations seem to be located in genes operating in the Krebs or glyoxylate cycle.