Dataset Information

Cytochrome b? coexpression increases Tetrahymena thermophila ?6 fatty acid desaturase activity in Saccharomyces cerevisiae.

ABSTRACT: Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on ?6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a ?6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2(?9,12) substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b? protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b? domains. Desaturation by the ?6 enzyme increased as much as 10-fold when T. thermophila cytochrome b?s were coexpressed with the desaturase. Coexpression of a cytochrome b? from Arabidopsis thaliana with the ?6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b? proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity.

SUBMITTER: Dahmen JL

PROVIDER: S-EPMC3675999 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Cytochrome b₅ coexpression increases Tetrahymena thermophila Δ6 fatty acid desaturase activity in Saccharomyces cerevisiae.

Dahmen Jeremy L JL Olsen Rebecca R Fahy Deirdre D Wallis James G JG Browse John J

Eukaryotic cell 20130412 6

Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was ...[more]

PMID: 23584993

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Project description:In response to a decrease in its growth temperature, the protozoan Tetrahymena is known to increase the level of unsaturated fatty acids in its membrane phospholipids so as to maintain the correct physical state (fluidity) of the membranes. In this organism, synthesis of unsaturated fatty acids is initiated by delta 9 acyl-CoA desaturase. Our previous studies have shown that, during cold adaptation, the activity of microsomal palmitoyl- and stearoyl-CoA desaturase increases, reaching a maximal level at 2 h after a temperature down-shift to 15 degrees C. Two hypotheses have been proposed to explain this increase in desaturase activity: (1) self-regulation via a direct effect of reduced membrane fluidity, and (2) induction of desaturase mRNA. However, the precise mechanism is not clearly understood. In order to obtain further insight into the mechanism of regulation of the desaturase, we have isolated a gene that encodes delta 9 fatty acid desaturase from T. thermophila and examined its expression during cold adaptation. The nucleotide sequence indicates that the 1.4 kbp gene encodes a polypeptide of 292 amino acid residues which shows marked sequence similarity to delta 9 acyl-CoA desaturases from other sources, e.g. rat, mouse, Amblyomma americanum and Saccharomyces cerevisiae. This protein has three histidine-cluster motifs (one HXXXXH and two HXXHH), and two hydrophobic regions which are conserved among delta 9 acyl-CoA desaturases. The level of desaturase mRNA was sensitive to decreasing the temperature of the culture media, and was close to maximal immediately after the temperature was shifted down from 35 degrees C to 15 degrees C (0.8 degrees C/min). Thereafter, the amount of mRNA gradually decreased with time, but remained above the control level for at least 5 h. Furthermore, during the course of the cooling process to 15 degrees C, the increased expression of desaturase mRNA became evident at 27 degrees C. Nuclear run-on analysis and actinomycin D chase experiments revealed that the elevation of the mRNA level was due to increases in both transcription and mRNA stability. These results suggest that the enhanced desaturase activity is controlled, at least in part, at the transcriptional level.

Dataset Information

Cytochrome b? coexpression increases Tetrahymena thermophila ?6 fatty acid desaturase activity in Saccharomyces cerevisiae.

Publications

Cytochrome b₅ coexpression increases Tetrahymena thermophila Δ6 fatty acid desaturase activity in Saccharomyces cerevisiae.

Similar Datasets

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