Identification of the Gene and Characterization of the Activity of the Trans-Aconitate Methyltransferase From Saccharomyces Cerevisiae

Biochemistry. 2001 Nov 13;40(45):13699-709. doi: 10.1021/bi011380j.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cytoplasm / enzymology
  • Escherichia coli / enzymology
  • Gene Expression*
  • Genes, Fungal*
  • Hydrolysis
  • Methyltransferases / genetics*
  • Methyltransferases / metabolism
  • Molecular Sequence Data
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Methyltransferases
  • aconitate methyltransferase