Carboxylation of phenolic compounds

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Phenolic compounds represent one of the most common natural compounds and therefore are important substrates for microorganisms. Their aerobic metabolism involves oxygenases and oxygen as a cosubstrate. In the absence of oxygen completely different reactions and enzymes are required. In the model bacteria Thauera aromatica and Azoarcus sp. EbN1, whose genome has been sequenced, phenol is first phosphorylated and then carboxylated yielding 4- hydroxybenzoate. This proposal has two aims. (1) The new enzymes of anaerobic phenol metabolism, phenylphosphate synthase and phenylphosphate carboxylase, will be studied in detail. In both cases enzyme-bound intermediates are to be expected, i.e. enzyme-phosphate and enzyme-phenolate, respectively. Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO2 as substrate, do not contain biotin or thiamine diphosphate, require K+ and a divalent metal cation (Mg2+or Mn2+) for activity, and are rapidly inactivated by oxygen. (2) The seven genes coding for these two enzymes are located on a large phenol induced operon which contains eight additional genes of unknown function. Lateron, it will be testet whether they are involved in the metabolism of related phenolic compounds. The carboxylation of various phenolic compounds such as catechol or ortho-cresol and the further metabolism of phenolic acids such as protocatechuate, vanillate and gentisate will be investigated. The results promise to provide insights into novel reactions and enzymes, to contribute to the understanding of the transformations of phenolic compounds in anoxic habitats notably in ground water and intestinal tract, and to lead to the biotechnological application of the carboxylation systems.