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

Xenobiotic phthalates are industrially produced on the annual million ton scale. The oxygen‐independent enzymatic reactions involved in anaerobic phthalate degradation have only recently been elucidated. In vitro assays suggested that phthalate is first activated to phthaloyl‐CoA followed by decarboxylation to benzoyl‐CoA. Here, we report the heterologous production and characterization of the enzyme initiating anaerobic phthalate degradation from ‘Aromatoleum aromaticum’: a highly specific succinyl‐CoA:phthalate CoA transferase (SPT, class III CoA transferase). Phthaloyl‐CoA formed by SPT accumulated only to sub‐micromolar concentrations due to the extreme lability of the product towards intramolecular substitution with a half‐life of around 7 min. Upon addition of excess phthaloyl‐CoA decarboxylase (PCD), the combined activity of both enzymes was drastically shifted towards physiologically relevant benzoyl‐CoA formation. In conclusion, a massive overproduction of PCD in phthalate‐grown cells to concentrations >140 μM was observed that allowed for efficient phthaloyl‐CoA conversion at concentrations 250‐fold below the apparent Km‐value of PCD. The results obtained provide insights into an only recently evolved xenobiotic degradation pathway where a massive cellular overproduction of PCD compensates for the formation of the probably most unstable CoA ester intermediate in biology.