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

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 s factor. This enzyme catalyzes the monomethyl esterification of transaconitate at high affinity (Km 5 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 Sadenosyl-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 2 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. Our laboratory has been interested in the protein L-isoaspartate (D-aspartate) O-methyltransferase (EC 2.1.1.77), an enzyme that catalyzes the methyl esterification of spontaneously altered residues in a pathway that can lead to the conversion of isomerized aspartyl residues to normal aspartyl residues in a net repair reaction (1). We have postulated that the physiological role of this methyltransferase is to preserve the integrity of the polypeptide chain in the face of age-dependent non-enzymatic reactions that lead to alterations in its configuration (2, 3). In the bacterium Escherichia coli, this enzyme is required for optimal survival of stationary phase cells against environmental stresses (4, 5), presumably functioning to maintain proteins in active configurations under conditions where protein synthesis to replace damaged proteins is limited. In the course of our studies of the protein L-isoaspartate methyltransferase encoded by the pcm gene in E. coli, we found an activity in cytosolic extracts that appeared to catalyze methyl ester formation but was not dependent upon the pcm gene product. We have now traced this activity to that of a previously undescribed small molecule methyltransferase that is active on trans-aconitate, an apparently non-enzymatically formed derivative of the citric acid cycle intermediate cisaconitate. Since both types of methyltransferase activities are directed to substrates that can be formed by spontaneous agerelated processes, we were interested in characterizing the trans-aconitate methyltransferase. We found that the expression of this activity is dependent on the stationary phase specific s factor RpoS. We have purified and characterized this enzyme, identified its gene, and characterized a knock-out strain lacking this activity. We were able to show that transaconitate is an endogenous substrate for the enzyme and that the product is its monomethyl ester.