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 Km of 0.66 mM. Other substrates were recognized much more poorly, including cis-aconitate with a Km of 74 mM and the decarboxylation product itaconate with a Km of 44 mM. The ratio of the maximal velocity to the Km 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-3H]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.
Cai
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Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, California 90095-1569, USA.
Dumlao
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D
Katz
J E
JE
Clarke
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D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005800:Genes, Fungal; D006868:Hydrolysis; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D008780:Methyltransferases; C119073:aconitate methyltransferase