An unusual strategy for the anoxic biodegradation of phthalate

Abstract

In the past two decades, the study of oxygen-independent degradation of widely abundant aromatic compounds in anaerobic bacteria has revealed numerous unprecedented enzymatic principles. Surprisingly, the organisms, metabolites and enzymes involved in the degradation of o-phthalate (1,2-dicarboxybenzene), mainly derived from phthalate esters that are annually produced at the million ton scale, are sparsely known. Here, we demonstrate a previously unknown capacity of complete phthalate degradation in established aromatic compound-degrading, denitrifying model organisms of the genera Thauera, Azoarcus and 'Aromatoleum'. Differential proteome analyses revealed phthalate-induced gene clusters involved in uptake and conversion of phthalate to the central intermediate benzoyl-CoA. Enzyme assays provided in vitro evidence for the formation of phthaloyl-CoA by a succinyl-CoA-and phthalate-specific CoA transferase, which is essential for the subsequent oxygen-sensitive decarboxylation to benzoyl-CoA. The extreme instability of the phthaloyl-CoA intermediate requires highly balanced CoA transferase and decarboxylase activities to avoid its cellular accumulation. Phylogenetic analysis revealed phthaloyl-CoA decarboxylase as a novel member of the UbiD-like, (de)carboxylase enzyme family. Homologs of the encoding gene form a phylogenetic cluster and are found in soil, freshwater and marine bacteria; an ongoing global distribution of a possibly only recently evolved degradation pathway is suggested.

Phthaloyl‐coenzyme A decarboxylase from Thauera chlorobenzoica: the prenylated flavin‐, K+‐ and Fe2+‐dependent key enzyme of anaerobic phthalate degradation

“…With 21 nmol min −1 mg −1 , the resulting specific activity in cell extracts is in the range of dearomatizing benzoyl‐CoA reductase (20–60 nmol min −1 mg −1 ) (Boll and Fuchs, ; Kuntze et al ., ; Tiedt et al ., ), the next enzyme in phthalate catabolism which is usually considered a bottleneck in the anaerobic catabolism of aromatic compounds. Taken into account the threefold higher doubling time during growth with phthalate vs. benzoate (Ebenau‐Jehle et al ., ), the activities determined in this work are in agreement with the observed growth parameters. Unexpectedly, the activity of PCD was higher in the presence of phthaloyl‐CoA forming CoA transferase plus their substrates than with chemically synthesized phthaloyl‐CoA.…”

“…In agreement with previous cell extract activity determinations, purified PCD was oxygen‐sensitive with a half‐life in air of 13 ± 3 min (Supporting Information Fig. S3) (Ebenau‐Jehle et al ., ). No change in hexameric subunit composition was observed upon oxygen‐exposure.…”

“…When anaerobically incubated enzyme was desalted in KCl‐free buffer a similar loss of activity and prFMN was observed as during incubation in air (Table ). This finding is in line with previous work, where a protecting effect of K + toward oxygen‐damage was reported (Ebenau‐Jehle et al ., ).…”

“…S1C and D). In agreement with earlier cell extract assays, 2‐cyanobenzoyl‐CoA, 2‐nitrobenzoyl‐CoA, 2‐methylphthaloyl‐CoA and maleoyl‐CoA were virtually not converted by purified PCD (<2% of the rate obtained with phthaloyl‐CoA) (Ebenau‐Jehle et al ., ).…”

Enzymes involved in phthalate degradation in sulphate‐reducing bacteria

“…The phthaloyl‐CoA forming and decarboxylating activities in D. cetonica crude extracts were in the range of the corresponding activities reported for extracts of denitrifying phthalate degraders (18–31 nmol min −1 mg −1 for succinyl‐CoA:phthalate CoA transferase and 6–13 nmol min −1 mg −1 for phthaloyl‐CoA decarboxylase activities) (Ebenau‐Jehle et al ., ).…”