Purification and Characterization of α-Keto Amide Reductase from<i>Saccharomyces cerevisiae</i>

Ishihara, Kohji; Yamamoto, Hiroaki; Mitsuhashi, Kazuya; Nishikawa, K.; Tsuboi, Sadao; Tsuji, Hideaki; Nakajima, Nobuyoshi

doi:10.1271/bbb.68.2306

Cited by 22 publications

(7 citation statements)

References 22 publications

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“…Three of the seven relatively minor luminal components of this category that were identified, are the translation products of ORFs YDL124w, YLR179c and YNL134c which have been tentatively identified as hypothetical proteins with α‐keto amide reductase activity [45] and as proteins bearing a resemblance to phosphatidylethanolamine binding and alcohol dehydrogenase proteins from other model organisms (SGD), respectively. The remaining four proteins are two about which nothing is known, YER004w and YHR029c, and two others, NCP1, an ER‐localized NADPH‐cytochrome P450 reductase and MCR1, cytochrome b5 reductase, a nuclearly encoded mitochondrial protein implicated in antimycin‐insensitive NADH oxidation [46].…”

Section: Resultsmentioning

confidence: 99%

Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae

Sarry¹,

Chen²,

Collum³

et al. 2007

The FEBS Journal

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Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.

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Section: Resultsmentioning

confidence: 99%

Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae

Sarry¹,

Chen²,

Collum³

et al. 2007

The FEBS Journal

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“…The functions of the remaining ORFs identified in our BLAST analysis, including YDL124W and YJR096W, have not been extensively studied. Ishihara recently reported evidence that a yeast protein, identical to that encoded by YDL124W, catalyzes the NADPH-dependent reduction of aromatic α-keto amides and α-keto esters [29]. Recent studies also indicate that the proteins encoded by YJR096W and YDL124W and may play a role in arabinose and xylose metabolism [30].…”

Section: Discussionmentioning

confidence: 99%

Functional studies of aldo-keto reductases in Saccharomyces cerevisiae

Chang

Griest

Harter

et al. 2007

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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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.

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“…Among the most upregulated genes in both strains upon furfural increase from 15 to 25 mM were the oxireductases ADH7, GRE2, OYE3, AAD4, YML131W, YDL124W, OYE2, and GCY1 (see Table S1 in the supplemental material). Six of them prefer NADPH as the cofactor (6,7,21,26,33,44), while for AAD4 and YML131W, the cofactor preference is not known (Saccharomyces Genome Database at http://www.yeastgenome.org). This is consistent with our finding that central carbon metabolism doubles the NADPH-generating pentose phosphate pathway fluxes upon 25 mM furfural challenge.…”

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confidence: 99%

Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases

Heer

Heine

Sauer

2009

Appl Environ Microbiol

147

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Biofuels derived from lignocellulosic biomass hold promises for a sustainable fuel economy, but several problems hamper their economical feasibility. One important problem is the presence of toxic compounds in processed lignocellulosic hydrolysates, with furfural as a key toxin. While Saccharomyces cerevisiae has some intrinsic ability to reduce furfural to the less-toxic furfuryl alcohol, higher resistance is necessary for process conditions. By comparing an evolved, furfural-resistant strain and its parent in microaerobic, glucose-limited chemostats at increasing furfural challenge, we elucidate key mechanism and the molecular basis of both natural and high-level furfural resistance. At lower concentrations of furfural, NADH-dependent oxireductases are the main defense mechanism. At furfural concentrations above 15 mM, however, 13 C-flux and global array-based transcript analysis demonstrated that the NADPH-generating flux through the pentose phosphate pathway increases and that NADPH-dependent oxireductases become the major resistance mechanism. The transcript analysis further revealed that iron transmembrane transport is upregulated in response to furfural. While these responses occur in both strains, high-level resistance in the evolved strain was based on strong induction of ADH7, the uncharacterized open reading frame (ORF) YKL071W, and four further, likely NADPHdependent, oxireductases. By overexpressing the ADH7 gene and the ORF YKL071W, we inversely engineered significantly increased furfural resistance in the parent strain, thereby demonstrating that these two enzymes are key elements of the resistance phenotype.

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Purification and Characterization of α-Keto Amide Reductase fromSaccharomyces cerevisiae

Cited by 22 publications

References 22 publications

Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae

Analysis of the vacuolar luminal proteome of Saccharomyces cerevisiae

Functional studies of aldo-keto reductases in Saccharomyces cerevisiae

Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases

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