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

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. The conversion of citrate to isocitrate catalyzed by aconitase in the tricarboxylic acid cycle of almost all organisms involves the intermediate formation of cis- aconitate. This compound is generally not released from the enzyme complex, but when it is, it can rebind and undergo the transformation into isocitrate ( 1). cis-Aconitate is not chemically stable and is readily converted to the more stable trans-aconitate form (2-5), which has been shown to be an effective inhibitor of aconitase from a number of organisms (6-10). It is unclear how cells prevent the accumulation of this potentially toxic metabolite. We previously identified a novel methyltransferase that esterifies trans-aconitate in Escherichia coli and have shown that it is encoded by the tam gene (11). This activity has also been detected in the yeast Saccharomyces cereVisiae although its structural gene was not identified ( 11). These activities can potentially result in the further metabolism of trans-aconitate, either by forming noninhibitory complexes or by potentially catalyzing one step in the conversion back to cis-aconitate. To begin to understand the metabolism of trans-aconitate by the methylation pathway, we first focused on determining the exact products of the reactions. It had not been established which of the three distinct carboxyl groups are modified and whether other covalent changes, such as a possible trans-cis isomerization may also ac- company the enzymatic reaction. trans-Aconitate can be potentially methyl-esterified to make one trimethyl, three structurally distinct dimethyl, and three structurally distinct monomethyl esters of trans- aconitate. We previously determined that the E. coli Tam 1 enzyme catalyzes the formation of monomethyl ester(s) by mass spectrometry (11). Enzymatically methylated trans- aconitate was found to coelute with only one of the chemically synthesized monomethyl esters, suggesting that methylation occurs on a specific site in trans-aconitate (11). Tandem mass spectrometry did not provide useful informa- tion on which of the three carboxylic acids were methylated by Tam due to the facile cleavage of the ester bond, and the initial loss of the methyl group in the fragmentation of the molecule. In this report, we have now used an advanced NMR technique to show that E. coli Tam catalyzes the formation of the 6-methyl ester of trans-aconitate. We have combined synthetic, NMR, mass spectrometric, and chro- matographic methods to also show that the yeast enzyme monomethylates trans-aconitate but on a distinct site to form the 5-methyl ester. These results show that a trans-cis isomerization does not accompany the methylation reaction and that the function of the methyltransferase in bacterial and lower eucaryotic cells may be distinct. Significantly, we have also shown that the methylesteri- fication of trans-aconitate largely relieves the inhibition of this compound on aconitase activity in both E. coli and yeast †