Reply to Behrman: “N-Formylmaleamic acid: An intermediate in nicotinic acid metabolism”

In reply to the letter from Behrman (1), we want to clarify the following. We have shown that hydrolysis of the N-formylmaleamic acid generated by the ring cleavage of 2,5-dihydroxypyridine by a dioxygenase enzyme (NicX) in Pseudomonas putida KT2440 is carried out by a dedicated deformylase enzyme (NicD) (2) and is not due to an additional activity of the dioxygenase enzyme, as was previously suggested (3). Although it had been shown earlier that N-formylmaleamic acid can undergo nonenzymatic hydrolysis and that this might explain why this compound was not detected by Gauthier and Rittenberg (3), under our assay conditions the N-formylmaleamic acid generated by NicX was readily detected, thereby rendering unlikely the possibility of nonenzymatic hydrolysis as the primary mechanism of decarboxylation. On the basis of these results, we suggested in our article that the previously implied dioxygenase-mediated decarboxylation of N-formylmaleamic acid can be now explained as a result of a deformylase activity that contaminated the purified dioxygenase (2). It is of course well known that sequentially acting enzymes sometimes copurify, and, indeed, the possibility of a minor contamination in the dioxygenase preparation was allowed by Gauthier and Rittenberg to explain their own results, and the lack of N-formylmaleamic acid hydrolysis observed (3) could be due to the fact that, according to Behrman, they might have used N-formylfumaramic instead of N-formylmaleamic acid as substrate (4). We do not therefore consider that we have misstated conclusions or impugned previous work, as indicated by Behrman (1), but simply have eliminated an earlier hypothesis and demonstrated that the hydrolysis of N-formylmaleamic acid in P. putida is not a dioxygenase-mediated reaction or a nonenzymatic process but rather a NicD-catalyzed reaction.