We have identified the yeast open reading frame YER175c as the gene encoding the trans-aconitate methyltransferase of Saccharomyces cerevisiae. Extracts of a yeast strain with a disrupted YER175c gene demonstrate a complete loss of activity toward the methyl-accepting substrates trans-aconitate, cis-aconitate, DL-isocitrate, and citrate. Reintroduction of the YER175c gene on a plasmid results in an overexpression of the activity toward each of these methyl-accepting substrates. We now designate this gene TMT1 for trans-aconitate methyltransferase. We examined the methyl-accepting substrate specificity of this enzyme in extracts from overproducing cells. We found that trans-aconitate was the best substrate with a K(m) of 0.66 mM. Other substrates were recognized much more poorly, including cis-aconitate with a K(m) of 74 mM and the decarboxylation product itaconate with a K(m) of 44 mM. The ratio of the maximal velocity to the K(m) of these substrates was only 0.24% and 0.9% that of trans-aconitate; for other substrates including citrate and other tricarboxylate and dicarboxylate derivatives, this ratio ranged from 0.0003% to 0.062% that of trans-aconitate. We then asked if any of these compounds were present endogenously in yeast extracts. We were able to identify trans-aconitate 5-methyl ester as well as additional unidentified radiolabeled products when S-adenosyl-L-[methyl-(3)H]methionine was mixed with TMT1(+) extracts (but not with tmt1(-) extracts), suggesting that there may be additional substrates for this enzyme. We showed that the product 5-methyl ester of trans-aconitate is not readily metabolized in yeast extracts. Finally, we demonstrated that the activity of the yeast trans-aconitate methyltransferase is localized in the cytosol and increases markedly as cells undergo the metabolic transition at the diauxic shift.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Reference||Annotation Extension|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Conditions||Strain||Source||Reference|