Abstract

In microbial degradation of hydroxy-aromatic compounds, preparation of the ring for cleavage is a crucial step. Two enzymes types involved in this are hydroxylases and decarboxylases. Hydroxylases introduce an oxygen atom into the aromatic ring while decarboxylases remove a carboxyl group. Bacteria and fungi are known to elaborate these types of enzymes but fungal forms have so-far received relatively little attention.
A. niger is a fungus which has been shown previously to grow on salicylate, benzoate, and several other aromatics1. The A. niger genome has many genes annotated as coding for aromatic hydroxylases and some decarboxylases: fifteen such genes were chosen for recombinant expression in E. coli. Of these, four corresponding hydroxylases were successfully expressed, purified, and characterized according to substrate specificity, cofactor requirement and quaternary structure. These proteins, all FAD-containing, comprise a true salicylate hydroxylase, two salicylate hydroxylase-like enzymes, and a hydroxylase active on p-cresol, resorcinol, and hydroquinone. While the first enzyme is likely to be involved in metabolism of salicylate, the functions of the two salicylate hydroxylase-like enzymes are unclear, as is the function of the p-cresol active enzyme. A fifth enzyme, 2,3-dihydroxybenzoate decarboxylase, was also successfully expressed in E. coli and purified. Although its properties were similar to those previously reported for the enzyme isolated from A. niger 2,3,4, the pH optimum appeared to be somewhat higher. In addition, it was found that this enzyme could catalyze the reverse reaction, carboxylation of catechol, and that after treatment with a mixture of chelators Ca2+ appeared to stimulate the activity