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Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

ABSTRACT: A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.

SUBMITTER: Bostian KA

PROVIDER: S-EPMC1185843 | biostudies-other | 1978 Sep

REPOSITORIES: biostudies-other

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Kinetics and reaction mechanism of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

Project description:Data from steady-state kinetic analysis of yeast K+-activated aldehyde dehydrogenase are consistent with a ternary complex mechanism. Evidence from alternative substrate analysis and product-inhibition studies supports an ordered sequence of substrate binding in which NAD+ is the leading substrate. A preincubation requirement for NAD+ for maximum activity is also consistent with the importance of a binary enzyme-NAD+ complex. Dissociation constant for enzyme-NAD+ complex determined kinetically is in reasonable agreement with that determined by direct binding. The order of substrate addition proposed here differs from that proposed for a yeast aldehyde dehydrogenase previously reported. Different methods of purification produced an enzyme that showed similar kinetic characteristics to those reported here.

| S-EPMC1185844 | biostudies-other

The purification and some properties of the Mg(2+)-activated cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae.

Project description:A purification procedure has been developed for the cytosolic aldehyde dehydrogenase of Saccharomyces cerevisiae that yields homogeneous enzyme. The enzyme seems to be a tetramer of identical 58 kDa subunits. The enzyme reaction is strongly stimulated by Mg2+ at low NADP+ concentrations but there is no absolute requirement for bivalent cations. The kinetics of the reaction have been studied in the presence and absence of MgCl2. NADP+ binding studies of the quenching of protein fluorescence in the presence and absence of MgCl2 show that the effect of Mg2+ is to increase the affinity of the enzyme for NADP+ by approx. 100-fold. NADP+ binding causes a slow conformational change in the enzyme and converts the enzyme from the inactive or low-activity form in which it is isolated into the fully active form. This conformational change seems to explain the marked lag-phases seen in enzyme assays. The enzyme is strongly inhibited by disulfiram and pyridoxal 5-phosphate.

| S-EPMC1217208 | biostudies-other

Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

Project description:Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC.

| S-EPMC4834435 | biostudies-literature

Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae.

Project description:When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast.

| S-EPMC4021361 | biostudies-literature

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae.

Project description:The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4%) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was studied. An enzyme group with pKa approx. 7.1 apparently promotes enzyme activity when in its alkaline form.

| S-EPMC1148419 | biostudies-other

Heterologous Expression of Aldehyde Dehydrogenase from Saccharomyces cerevisiae in Klebsiella pneumoniae for 3-Hydroxypropionic Acid Production from Glycerol.

Project description:3-Hydroxypropionic acid (3-HP) is a commercially valuable platform compound. Klebsiella pneumoniae has been concerned as an appropriate host for 3-HP production because of its robust capacity to metabolize glycerol. Glycerol conversion to 3-HP in K. pneumoniae comprises two successive reactions: glycerol dehydratase catalyzes glycerol to 3-hydroxypropionaldehyde (3-HPA); aldehyde dehydrogenase catalyzes 3-HPA to 3-HP. Previous studies focusing on inducible expression of aldehyde dehydrogenase have shown defects of high cost of inducer and low catalytic activity due to inclusion body. Here we show a different strategy that a native promoter in the host K. pneumoniae was used to drive the heterologous expression of aldehyde dehydrogenase gene ald4 from Saccharomyces cerevisiae. The 3-HP yield of the recombinant reached a peak of 4.23 g/L at log phase, but it decreased during later period of fermentation. Except the validation of high activity of ald4, particularly, the 3-HP formation was uncovered to be closely coupled with cell division, and the lacking of NAD and ATP at latter fermentation phase became the bottleneck for cell growth and 3-HP accumulation. Furthermore, 3-HP is postulated to be converted to 3-HPA via feedback inhibition or other metabolite via unknown mechanism. Since glycerol dissimilation is a common mechanism in a variety of bacteria, the expression strategy using native promoter and implications may provide significant insight into the metabolic engineering for 3-HP production.

| S-EPMC3460132 | biostudies-literature

Transcriptional response of Saccharomyces cerevisiae to potassium starvation.

Project description:BACKGROUND: Ion homeostasis is essential for every cell and aberrant cation homeostasis is related to diseases like Alzheimer's disease and epilepsy. The mechanisms responsible for cation homeostasis are only partly understood. The yeast Saccharomyces cerevisiae is an excellent organism to study fundamental aspects of cation homeostasis. In this study we investigated the transcriptional response of this yeast to potassium starvation by using Serial Analysis of Gene Expression (SAGE)-tag sequencing. RESULTS: Comparison of transcript levels in cells grown for 60 min in media without potassium with those in cells grown under standard potassium concentrations showed that the mRNA levels of 105 genes were significantly (P < 0.01) up-regulated more than 2.0-fold during potassium starvation and the mRNA levels of 172 genes significantly down-regulated. These genes belong to several functional categories. Genes involved in stress response including HSP30, YRO2 and TPO2 and phosphate metabolism including PHO84, PHO5 and SPL2 were highly up-regulated. Analysis of the promoter of PHO84 encoding a high affinity phosphate transporter, revealed that increased PHO84 RNA levels are caused by both increased Pho4-dependent transcription and decreased RNA turnover. In the latter process antisense transcription may be involved. Many genes involved in cell cycle control, and to a lesser extent genes involved in amino acid transport, were strongly down-regulated. CONCLUSIONS: Our study showed that yeast cells respond to potassium starvation in a complex way and reveals a direct link between potassium homeostasis and phosphate metabolism.

| S-EPMC4289377 | biostudies-literature

Purification and characterization of Put1p from Saccharomyces cerevisiae.

Project description:In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A K(m) value of 36 mM proline and a k(cat)=27 s(-1) were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ(1)) as an electron acceptor with a k(cat)=9.6 s(-1) and a K(m) of 33 microM for CoQ(1). In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast.

| S-EPMC2880193 | biostudies-literature

Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii.

Project description:Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met177, compared with the equivalent residue Leu196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity.

| S-EPMC5483954 | biostudies-literature

Molecular cloning of the mitochondrial aldehyde dehydrogenase gene of Saccharomyces cerevisiae by genetic complementation.

Project description:Mutants of Saccharomyces cerevisiae deficient in mitochondrial aldehyde dehydrogenase (ALDH) activity were isolated by chemical mutagenesis with ethyl methanesulfonate. The mutants were selected by their inability to grow on ethanol as the sole carbon source. The ALDH mutants were distinguished from alcohol dehydrogenase mutants by an aldehyde indicator plate test and by immunoscreening. The ALDH gene was isolated from a yeast genomic DNA library on a 5.7-kb insert of a recombinant DNA plasmid by functional complementation of the aldh mutation in S. cerevisiae. An open reading frame which specifies 533 codons was found within the 2.0-kb BamHI-BstEII fragment in the 5.7-kb genomic insert which can encode a protein with a molecular weight of 58,630. The N-terminal portion of the protein contains many positively charged residues which may serve as a signal sequence that targets the protein to the mitochondria. The amino acid sequence of the proposed mature yeast enzyme shows 30% identity to each of the known ALDH sequences from eukaryotes or prokaryotes. The amino acid residues corresponding to mammalian cysteine 302 and glutamates 268 and 487, implicated to be involved at the active site, were conserved. S. cerevisiae ALDH was found to be localized in the mitochondria as a tetrameric enzyme. Thus, that organelle is responsible for acetaldehyde oxidation, as was found in mammalian liver.

| S-EPMC207915 | biostudies-other

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