Possible radical mechanism in enzymatic carboxylation of phenol

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Anaerobic metabolism of phenol proceeds via para carboxylation to 4-hydroxybenzoate. This endergonic reaction is known since long in chemistry (Kolbe-Schmitt-carboxylation), but is only poorly studied in biology. The phenol carboxylating enzyme system in the bacterium Thauera aromatica will be investigated. It does not belong to any group of the studied carboxylases as it proceeds in two steps via a phosphorylated free intermediate (phenylphosphate) and uses a metal (Mn2+) as co-catalyst and carbon dioxide as substrate. While phosphorylation of phenol seems essential to drive carboxylation forward, it makes phenylphosphate a poor substrate for CO2 attack. The phenol carboxylating system does not depend on biotin or thiamine diphosphate and is complex; 10 proteins are phenol-induced. The carboxylase belongs to a family of proteins whose functions are largely unknown. Most remarkably, the enzyme is extremely sensitive to oxygen and radical trapping agents. The phenyl-phosphate carboxylating enzyme will be purified and characterized. Special focus will be on the catalytic mechanism which according to our working hypothesis involves a strongly enzyme-bound phenolate intermediate and a radical species.