Publications

• A novel methyltransferase catalyzes the methyl esterification of trans-aconitate in Escherichia coli.

  Cai H., Clarke S.

  We have identified a new type of S-adenosyl-L-methionine-dependent methyltransferase in the cytosol of Escherichia coli that is expressed in early stationary phase under the control of the RpoS sigma factor. This enzyme catalyzes the monomethyl esterification of trans-aconitate at high affinity (K ... >> More

  We have identified a new type of S-adenosyl-L-methionine-dependent methyltransferase in the cytosol of Escherichia coli that is expressed in early stationary phase under the control of the RpoS sigma factor. This enzyme catalyzes the monomethyl esterification of trans-aconitate at high affinity (Km = 0.32 mM) and cis-aconitate, isocitrate, and citrate at lower velocities and affinities. We have purified the enzyme to homogeneity by gel-filtration, anion-exchange, and hydrophobic chromatography. The N-terminal amino acid sequence was found to match that expected for the o252 open reading frame at 34.57 min on the E. coli genomic sequence whose deduced amino acid sequence contains the signature sequence motifs of the major class of S-adenosyl-L-methionine-dependent methyltransferases. Overexpression of the o252 gene resulted in an overexpression of the methyltransferase activity, and we have now designated it tam for trans-aconitate methyltransferase. We have generated a knock-out strain of E. coli lacking this activity, and we find that its growth and stationary phase survival are similar to that of the parent strain. We demonstrate the endogenous formation of trans-aconitate methyl ester in extracts of wild type but not tam-mutant cells indicating that trans-aconitate is present in E. coli. Since trans-aconitate does not appear to be a metabolic intermediate in these cells but forms spontaneously from the key citric acid cycle intermediate cis-aconitate, we suggest that its methylation may limit its potential interference in normal metabolic pathways. We have detected trans-aconitate methyltransferase activity in extracts of the yeast Saccharomyces cerevisiae, whereas no activity has been found in extracts of Caenorhabditis elegans or mouse brain. << Less

  J. Biol. Chem. 274:13470-13479(1999) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Distinct reactions catalyzed by bacterial and yeast trans-aconitate methyltransferases.

  Cai H., Strouse J., Dumlao D., Jung M.E., Clarke S.

  The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxy ... >> More

  The trans-aconitate methyltransferase from the bacterium Escherichia coli catalyzes the monomethyl esterification of trans-aconitate and related compounds. Using two-dimensional (1)H/(13)C nuclear magnetic resonance spectroscopy, we show that the methylation is specific to one of the three carboxyl groups and further demonstrate that the product is the 6-methyl ester of trans-aconitate (E-3-carboxy-2-pentenedioate 6-methyl ester). A similar enzymatic activity is present in the yeast Saccharomyces cerevisiae. Although we find that yeast trans-aconitate methyltransferase also catalyzes the monomethyl esterification of trans-aconitate, we identify that the methylation product of yeast is the 5-methyl ester (E-3-carboxyl-2-pentenedioate 5-methyl ester). The difference in the reaction catalyzed by the two enzymes may explain why a close homologue of the E. coli methyltransferase gene is not found in the yeast genome and furthermore suggests that these two enzymes may play distinct roles. However, we demonstrate here that the conversion of trans-aconitate to each of these products can mitigate its inhibitory effect on aconitase, a key enzyme of the citric acid cycle, suggesting that these methyltransferases may achieve the same physiological function with distinct chemistries. << Less

  Biochemistry 40:2210-2219(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

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

  Cai H., Dumlao D.S., Katz J.E., Clarke S.

  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, c ... >> More

  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. << Less

  Biochemistry 40:13699-13709(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• 3-isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase.

  Katz J.E., Dumlao D.S., Wasserman J.I., Lansdown M.G., Jung M.E., Faull K.F., Clarke S.

  The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may ... >> More

  The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may attenuate trans-aconitate inhibition of aconitase and other enzymes of the cycle. Although trans-aconitate is a minor endogenous substrate of the Tmt1 enzyme in extracts of S. cerevisiae, the major endogenous substrate has yet to be identified. We show here that a trimethylsilylated derivative of the major methylated endogenous product of Tmt1 in yeast extracts has an identical gas chromatography retention time and an identical electron impact mass spectrum as one of the two possible monomethyl ester derivatives of (2R,3S)-3-isopropylmalate. (2R,3S)-3-Isopropylmalate is an intermediate of the leucine biosynthetic pathway that shares similar intermediates and reaction chemistry with the portion of the citric acid cycle from oxaloacetate to alpha-ketoglutarate via cis-aconitate. The Tmt1 methyltransferase recognizes (2R,3S)-3-isopropylmalate with similar kinetics as it does trans-aconitate, with respective K(m) values of 127 and 53 microM and V(max) values of 59 and 70 nmol min(-1) mg(-1) of protein in a Tmt1-overexpressed yeast extract. However, we found that isopropylfumarate, the direct homologue of trans-aconitate in the leucine biosynthetic pathway, was at best a very poor substrate for the Tmt1 yeast enzyme. Similarly, the direct homologue of 3-isopropylmalate in the citric acid cycle, isocitrate, is also a very poor substrate. This apparent change in specificity between the intermediates of these two pathways can be understood in terms of the binding of these substrates to the active site. These results suggest that the Tmt1 methyltransferase may work in two different pathways in two different ways: for detoxification in the citric acid cycle and for a possibly novel biosynthetic branch reaction of the leucine biosynthetic pathway. << Less

  Biochemistry 43:5976-5986(2004) [PubMed] [EuropePMC]