Identification of the gene and characterization of the activity of the trans-aconitate methyltransferase from Saccharomyces cerevisiae

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 nM. 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 Km of these substrates was only 0.24% and 0.9% that of traps-aconitate; for other substrates including citrate and other tricarboxylate and dicarboxylate derivatives, this ratio ranged from 0.0003% to 0.062% that of traps-aconitate. We then asked if any of these compounds were present endogenously in yeast extracts. We were able to identify traps-aconitate 5-methyl ester as well as additional unidentified radiolabeled products when S-adenosyl-L-[methyl-H-3]methionine was mixed with TMT1(+) extracts (but not with tmtl(-) extracts), suggesting that there may be additional substrates for this enzyme. We showed that the product 5-methyl ester of traps-aconitate is not readily metabolized in yeast extracts. Finally, we demonstrated that the activity of the yeast traps-aconitate methyltransferase is localized in the cytosol and increases markedly as cells undergo the metabolic transition at the diauxic shift.