Phthaloyl‐coenzyme A decarboxylase from <i>Thauera chlorobenzoica</i> : the prenylated flavin‐, K ⁺ ‐ and Fe ²⁺ ‐dependent key enzyme of anaerobic phthalate degradation

Abstract

The degradation of the industrially produced and environmentally relevant phthalate esters by microorganisms is initiated by the hydrolysis to alcohols and phthalate (1,2-dicarboxybenzene). In the absence of oxygen the further degradation of phthalate proceeds via activation to phthaloyl-CoA followed by decarboxylation to benzoyl-CoA. Here, we report on the first purification and characterization of a phthaloyl-CoA decarboxylase (PCD) from the denitrifying Thauera chlorobenzoica. Hexameric PCD belongs to the UbiD-family of (de)carboxylases and contains prenylated FMN (prFMN), K⁺ and, unlike other UbiD-like enzymes, Fe²⁺ as cofactors. The latter is suggested to be involved in oxygen-independent electron-transfer during oxidative prFMN maturation. Either oxidation to the Fe³⁺ -state in air or removal of K⁺ by desalting resulted in >92% loss of both, prFMN and decarboxylation activity suggesting the presence of an active site prFMN/Fe²⁺ /K⁺ -complex in PCD. The PCD-catalysed reaction was essentially irreversible: neither carboxylation of benzoyl-CoA in the presence of 2 M bicarbonate, nor an isotope exchange of phthaloyl-CoA with ¹³ C-bicarbonate was observed. PCD differs in many aspects from prFMN-containing UbiD-like decarboxylases and serves as a biochemically accessible model for the large number of UbiD-like (de)carboxylases that play key roles in the anaerobic degradation of environmentally relevant aromatic pollutants.