Naphthalene Carboxylation in the Sulfate-Reducing Enrichment Culture N47 Is Proposed to Proceed via 1,3-Dipolar Cycloaddition to the Cofactor Prenylated Flavin Mononucleotide.

Abstract

Polycyclic aromatic hydrocarbons are persistent pollutants of anthropogenic or natural origin in the environment and accumulate in anoxic habitats. In this study, we investigated the mechanism of the enzyme naphthalene carboxylase as a model reaction for polycyclic aromatic hydrocarbon activation by carboxylation. An enzyme assay was established with cell extracts of the highly enriched culture N47. In assays without addition of ATP, naphthalene carboxylase catalyzed a stable isotope exchange of the carboxyl group of naphthoate with 13C-labeled bicarbonate buffer, which can only occur via a partial backwards reaction of the naphthalene carboxylase reaction to an intermediate that does not include the carboxyl group. Hence, a new carboxyl group from the labeled bicarbonate is added upon forward reaction to the naphthoate. This indicates that the reaction mechanism consists of two or more steps and that at least the latter steps are reversible and ATP independent. Naphthalene carboxylation assays were carried out in deuterated buffer and revealed the incorporation of 0, 1, 2, or 3 deuterium atoms in the final product naphthoyl-coenzyme A, indicating that the reaction is fully reversible. Putative reaction mechanisms were tested by quantum mechanical calculations. The proposed mechanism of the reaction consists of three steps: the activation of the naphthalene by 1,3-dipolar cycloaddition of the cofactor prFMN to naphthalene, release of a proton and rearomatization producing a stable intermediate, and a carboxylation with a reverse 1,3-dipolar cycloaddition and cleavage of the bond to the cofactor producing 2-naphthoate. IMPORTANCE Pollution with polycyclic aromatic hydrocarbons poses a great hazard to humans and animals, with considerable long-term effects. The anaerobic degradation of polycyclic aromatic hydrocarbons in anoxic zones and anaerobic growth of such organisms is very slow, leading to only poor investigation of the degradation pathways, so far. In this work, we elucidated the mechanism of naphthalene carboxylase, a key enzyme in anaerobic naphthalene degradation. This is the first mechanism proposed for a carboxylase targeting nonsubstituted (polycyclic) aromatic compounds and can serve as a model for the initial activation reaction in the anaerobic degradation of benzene or nonsubstituted polycyclic aromatic hydrocarbons, as well as similar enzymatic reactions from the expanding class of UbiD-like (de)carboxylases.