Purification and Properties of Dihydroxyacetone Kinase from Schizosaccharomyces pombe

Marshall, J. H.; May, John W.; Sloan, Joan; Vasiliadis, George

doi:10.1099/00221287-132-9-2611

Cited by 4 publications

(2 citation statements)

References 11 publications

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“…This was clearly substantiated for the DAK1-encoded protein, since a gene deletion abolished measurable DAK activity, whereas DAK1 overexpression led to radically higher levels of DAK activity during growth in glucose medium. In addition, the kinetic properties of the Dak1 protein in S. cerevisiae, with a here reported K m for DHA of 22 M and for ATP of 0.49 mM, are similar to most of the earlier charac- (26,28,34). These values for the yeast enzymes are also in good agreement with K m(DHA) of DAKs from diverse bacterial species (27).…”

Section: Discussionsupporting

confidence: 87%

Dihydroxyacetone Kinases in Saccharomyces cerevisiaeAre Involved in Detoxification of Dihydroxyacetone

Molin

Norbeck²,

Blomberg

2003

Journal of Biological Chemistry

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The genes YML070W/DAK1 and YFL053W/DAK2 in the yeast Saccharomyces cerevisiae were characterized by a combined genetic and biochemical approach that firmly functionally classified their encoded proteins as dihydroxyacetone kinases (DAKs), an enzyme present in most organisms. The kinetic properties of the two isoforms were similar, exhibiting K m(DHA) of 22 and 5 M and K m(ATP) of 0.5 and 0.1 mM for Dak1p and Dak2p, respectively. We furthermore show that their substrate, dihydroxyacetone (DHA), is toxic to yeast cells and that the detoxification is dependent on functional DAK. The importance of DAK was clearly apparent for cells where both isogenes were deleted (dak1⌬dak2⌬), since this strain was highly sensitive to DHA. In the opposite case, overexpression of either DAK1 or DAK2 made the dak1⌬dak2⌬ highly resistant to DHA. In fact, overexpression of either DAK provided cells with the capacity to grow efficiently on DHA as the only carbon and energy source, with a generation time of about 5 h. The DHA toxicity was shown to be strongly dependent on the carbon and energy source utilized, since glucose efficiently suppresses the lethality, whereas galactose or ethanol did so to a much lesser extent. However, this suppression was found not to be explained by differences in DHA uptake, since uptake kinetics revealed a simple diffusion mechanism with similar capacity independent of carbon source. Salt addition strongly aggravated the DHA toxicity, independent of carbon source. Furthermore, the DHA toxicity was not linked to the presence of oxygen or to the known harmful agents methylglyoxal and formaldehyde. It is proposed that detoxification of DHA may be a vital part of the physiological response during diverse stress conditions in many species.

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Section: Discussionsupporting

confidence: 87%

Dihydroxyacetone Kinases in Saccharomyces cerevisiaeAre Involved in Detoxification of Dihydroxyacetone

Molin

Norbeck²,

Blomberg

2003

Journal of Biological Chemistry

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“…On the other hand, Schizosaccharomyces pombe and a number of glycerol-utilizing yeasts were assumed to produce DHA phosphate via DHA on the basis of the observation of the absence of glycerol kinase and the presence of glycerol dehydrogenase and DHA kinase (DHAK) (11). Marshall et al (10) purified a DHAK from S. pombe 972h Ϫ which is a key enzyme in the main pathway of glycerol dissimilation and showed that its native form was a molecule composed of four identical subunits with a molecular mass of 45,000 Da. We have found that S. pombe IFO 0354 produces two immunologically distinct DHAKs, and we have characterized these enzymes.…”

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confidence: 99%

Purification and Characterization of Two Dihydroxyacetone Kinases from Schizosaccharomyces pombe IFO 0354

Yoshihara¹,

Shimada²,

Karita³

et al. 1996

Appl Environ Microbiol

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Two dihydroxyacetone kinases (DHAKs), DHAK I and DHAK II, were purified to homogeneity from Schizosaccharomyces pombe IFO 0354. They were immunologically different from each other. Although both of the enzymes had some affinity for glycerol and DL-glyceraldehyde in addition to dihydroxyacetone and glyceraldehyde, V max values for dihydroxyacetone were much higher than those for glycerol and DL-glyceraldehyde. On the basis of the K m values of both enzymes for dihydroxyacetone, DHAK II plays a more important role than DHAK I in dissimilation of glycerol via dihydroxyacetone.

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Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe , and its application to dihydroxyacetone phosphate production

Tujibata

Liu²

1999

Applied Microbiology and Biotechnology

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The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62,245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate.

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Purification and Properties of Dihydroxyacetone Kinase from Schizosaccharomyces pombe

Cited by 4 publications

References 11 publications

Dihydroxyacetone Kinases in Saccharomyces cerevisiaeAre Involved in Detoxification of Dihydroxyacetone

Dihydroxyacetone Kinases in Saccharomyces cerevisiaeAre Involved in Detoxification of Dihydroxyacetone

Purification and Characterization of Two Dihydroxyacetone Kinases from Schizosaccharomyces pombe IFO 0354

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe , and its application to dihydroxyacetone phosphate production

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