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Cloning and characterization of a sulfonate/alpha-ketoglutarate dioxygenase from Saccharomyces cerevisiae.

ABSTRACT: The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/alpha-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/alpha-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates being isethionate and taurocholate. Disruption of the gene encoding this enzyme negatively affects the use of isethionate and taurine as sulfur sources by S. cerevisiae, providing strong evidence that YLL057c plays a role in sulfonate catabolism.

SUBMITTER: Hogan DA

PROVIDER: S-EPMC94115 | biostudies-literature | 1999 Sep

REPOSITORIES: biostudies-literature

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Cloning and characterization of a sulfonate/alpha-ketoglutarate dioxygenase from Saccharomyces cerevisiae.

Hogan D A DA Auchtung T A TA Hausinger R P RP

Journal of bacteriology 19990901 18

The Saccharomyces cerevisiae open reading frame YLL057c is predicted to encode a gene product with 31.5% amino acid sequence identity to Escherichia coli taurine/alpha-ketoglutarate dioxygenase and 27% identity to Ralstonia eutropha TfdA, a herbicide-degrading enzyme. Purified recombinant yeast protein is shown to be an Fe(II)-dependent sulfonate/alpha-ketoglutarate dioxygenase. Although taurine is a poor substrate, a variety of other sulfonates are utilized, with the best natural substrates bei ...[more]

PMID: 10482536

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Project description:?-Ketoglutarate (?KG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae ?KG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (??5) showed that ?KG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of ?KG was further investigated under ?KG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated ?KG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of ?KG. Consumption of ?KG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative ?KG transporter is repressed by glucose. Fully 13C-labeled ?KG was introduced as a tracer during a glucose/?KG co-feeding chemostat to trace ?KG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C ?KG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular ?KG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on ?KG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of ?KG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic ?KG as precursor, both observed sinks should be minimized to increase the putative yields.

Project description:The full-length DNAs for two Saccharomyces cerevisiae aldehyde dehydrogenase (ALDH) genes were cloned and expressed in Escherichia coli. A 2,744-bp DNA fragment contained an open reading frame encoding cytosolic ALDH1, with 500 amino acids, which was located on chromosome XVI. A 2,661-bp DNA fragment contained an open reading frame encoding mitochondrial ALDH5, with 519 amino acids, of which the N-terminal 23 amino acids were identified as the putative leader sequence. The ALDH5 gene was located on chromosome V. The commercial ALDH (designated ALDH2) was partially sequenced and appears to be a mitochondrial enzyme encoded by a gene located on chromosome XV. The recombinant ALDH1 enzyme was found to be essentially NADP dependent, while the ALDH5 enzyme could utilize either NADP or NAD as a cofactor. The activity of ALDH1 was stimulated two- to fourfold by divalent cations but was unaffected by K+ ions. In contrast, the activity of ALDH5 increased in the presence of K+ ions: 15-fold with NADP and 40-fold with NAD, respectively. Activity staining of isoelectric focusing gels showed that cytosolic ALDH1 contributed 30 to 70% of the overall activity, depending on the cofactor used, while mitochondrial ALDH2 contributed the rest. Neither ALDH5 nor the other ALDH-like proteins identified from the genomic sequence contributed to the in vitro oxidation of acetaldehyde. To evaluate the physiological roles of these three ALDH isoenzymes, the genes encoding cytosolic ALDH1 and mitochondrial ALDH2 and ALDH5 were disrupted in the genome of strain TWY397 separately or simultaneously. The growth of single-disruption delta ald1 and delta ald2 strains on ethanol was marginally slower than that of the parent strain. The delta ald1 delta ald2 double-disruption strain failed to grow on glucose alone, but growth was restored by the addition of acetate, indicating that both ALDHs might catalyze the oxidation of acetaldehyde produced during fermentation. The double-disruption strain grew very slowly on ethanol. The role of mitochondrial ALDH5 in acetaldehyde metabolism has not been defined but appears to be unimportant.

Dataset Information

Cloning and characterization of a sulfonate/alpha-ketoglutarate dioxygenase from Saccharomyces cerevisiae.

Publications

Cloning and characterization of a sulfonate/alpha-ketoglutarate dioxygenase from Saccharomyces cerevisiae.

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