Enzymes involved in the anaerobic degradation of ortho‐phthalate by the nitrate‐reducing bacterium Azoarcus sp. strain PA01

Abstract

The pathway of anaerobic degradation of o-phthalate was studied in the nitrate-reducing bacterium Azoarcus sp. strain PA01. Differential two-dimensional protein gel profiling allowed the identification of specifically induced proteins in o-phthalate-grown compared to benzoate-grown cells. The genes encoding o-phthalate-induced proteins were found in a 9.9 kb gene cluster in the genome of Azoarcus sp. strain PA01. The o-phthalate-induced gene cluster codes for proteins homologous to a dicarboxylic acid transporter, putative CoA-transferases and a UbiD-like decarboxylase that were assigned to be specifically involved in the initial steps of anaerobic o-phthalate degradation. We propose that o-phthalate is first activated to o-phthalyl-CoA by a putative succinyl-CoA-dependent succinyl-CoA:o-phthalate CoA-transferase, and o-phthalyl-CoA is subsequently decarboxylated to benzoyl-CoA by a putative o-phthalyl-CoA decarboxylase. Results from in vitro enzyme assays with cell-free extracts of o-phthalate-grown cells demonstrated the formation of o-phthalyl-CoA from o-phthalate and succinyl-CoA as CoA donor, and its subsequent decarboxylation to benzoyl-CoA. The putative succinyl-CoA:o-phthalate CoA-transferase showed high substrate specificity for o-phthalate and did not accept isophthalate, terephthalate or 3-fluoro-o-phthalate whereas the putative o-phthalyl-CoA decarboxylase converted fluoro-o-phthalyl-CoA to fluoro-benzoyl-CoA. No decarboxylase activity was observed with isophthalyl-CoA or terephthalyl-CoA. Both enzyme activities were oxygen-insensitive and inducible only after growth with o-phthalate. Further degradation of benzoyl-CoA proceeds analogous to the well-established anaerobic benzoyl-CoA degradation pathway of nitrate-reducing bacteria.

Enzymes involved in phthalate degradation in sulphate‐reducing bacteria

“…This finding is in accordance with the absence of a gene encoding a putative succinyl‐CoA:phthalate CoA transferase in the phthalate induced gene cluster in sulphate‐reducing bacteria. In contrast, succinyl‐CoA represents a highly abundant CoA ester in phthalate‐degrading denitrifying bacteria and serves as CoA donor for a phthalate induced CoA transferase (Junghare et al ., ; Ebenau‐Jehle et al ., ; Mergelsberg et al ., ). The inability to detect phthaloyl‐CoA is assigned to its reported instability which after its formation affords an immediate complexation and decarboxylation by the highly abundant PCD, which prevents its accumulation and subsequent decay (Mergelsberg et al ., ).…”

“…PA01 activated phthalate to phthaloyl‐CoA by a succinyl‐CoA‐dependent CoA transferase, followed by decarboxylation to benzoyl‐CoA (Fig. ) (Junghare et al ., ; Ebenau‐Jehle et al ., ). In denitrifying bacteria, benzoyl‐CoA is dearomatized by ATP‐dependent class I benzoyl‐CoA reductases (Kung et al ., ; Boll et al ., ; Buckel et al ., ; Tiedt et al ., ).…”

Novel Three-Component Phenazine-1-Carboxylic Acid 1,2-Dioxygenase in Sphingomonas wittichii DP58

“…Native molecular weight of PcaA1A2 was measured using proteinfolding liquid chromatography (PFLC). Decarboxylation by different mechanisms has been found during the degradation of benzoic acid and substituted benzoate (27)(28)(29)(30). Using a conserved domain analysis, a ferredoxin-binding domain and AhdA1 c -like domain were found in the N and C termini, respectively, of PcaA1.…”

Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl‐CoA intermediate during anaerobic phthalate degradation

“…Using extracts from Thauera, Azoarcus and ‘ Aromatoleum’ strains that had been anaerobically grown with phthalate and nitrate, phthalate was first activated to phthaloyl‐CoA in a succinyl‐CoA dependent manner which was subsequently decarboxylated to benzoyl‐CoA (Fig. ), (Junghare et al ., ; Ebenau‐Jehle et al ., ). In these assays the proposed phthaloyl‐CoA intermediate was not detected by UPLC‐UV/vis analyses and its formation could only qualitatively be demonstrated by mass spectrometry (MS) (Ebenau‐Jehle et al ., ; Junghare et al ., ).…”