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A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster
Published on: January 16, 2020
Functional comparison of citrate synthase isoforms from S. cerevisiae
Eric R Graybill1, Matthew F Rouhier, Charles E Kirby
1Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056, USA.
Archives of Biochemistry and Biophysics
|June 16, 2007
Summary
This study identifies CIT3 as a dual citrate and methylcitrate synthase in yeast, crucial for propionate metabolism. Deleting CIT2 enables growth on propionate by preventing toxic methylcitrate buildup.
Area of Science:
- Biochemistry
- Molecular Biology
- Yeast Genetics
Background:
- Citrate synthases are key enzymes in the TCA cycle.
- Understanding propionate metabolism in Saccharomyces cerevisiae is important for industrial applications.
- The roles of specific citrate synthase isozymes, CIT1, CIT2, and CIT3, in yeast metabolism require further elucidation.
Purpose of the Study:
- To characterize the substrate specificities of CIT1 and CIT3 gene products.
- To investigate the roles of CIT1, CIT2, and CIT3 in propionate metabolism and methylcitrate production.
- To elucidate the metabolic fate of propionate in yeast mutants.
Main Methods:
- Recombinant protein expression and enzyme activity assays.
- Gene deletion mutant construction in Saccharomyces cerevisiae.
- 13C Nuclear Magnetic Resonance (NMR) and Gas Chromatography-Mass Spectrometry (GC-MS) analyses.
Main Results:
- CIT3 encodes a mitochondrial dual specificity citrate and methylcitrate synthase, while CIT1 is specific for citrate.
- Deletion of CIT2 allows yeast growth on propionate, indicating toxic methylcitrate accumulation in wild-type peroxisomes.
- CIT3 is essential for propionate metabolism, and its absence leads to altered pyruvate metabolism, including acetate and isobutanol accumulation.
Conclusions:
- CIT3 plays a critical role in both citrate and methylcitrate synthesis, essential for yeast growth on propionate.
- CIT2 is involved in the toxic accumulation of methylcitrate during propionate metabolism.
- Metabolic flux through the pyruvate dehydrogenase complex is affected by propionyl-CoA in CIT3-deficient mutants.
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