Abstract

Fungi, together with bacteria, are responsible for the degradation of aromatic compounds in the environment. The β-ketoadipate pathway is a common pathway for the cleavage of dihydroxylated aromatics such as catechol but while the bacterial enzymes for this pathway have been well-characterized, those from fungi have not.
This pathway in bacteria includes two branches: one with six enzymes to convert catechol to acetyl coenzyme A and succinate, and the other with seven enzymes to catalyze the conversion of protocatechuate (3,4-dihydroxybenzoate) to the same products plus CO2. BLAST sequence comparisons identified genes in the Aspergillus niger genome that are likely to encode these enzymes. Aspergillus niger cDNA was used to amplify 12 of these genes, which were subsequently cloned using ligation-independent cloning into the pLATE11 vector and the resulting plasmids were tested for expression in E.coli BL21. Of these, 4 proteins were successfully expressed and then purified using a combination of column chromatography techniques. Assays of two of these proteins combined with UV analysis of reaction intermediates indicated that they encode β-carboxymuconate cyclase (NRRL3_02586) and β-carboxymuconolactone hydrolase/decarboxylase (NRRL3_01409) activities from the protocatechuate branch of the pathway. The β-carboxymuconolactone hydrolase/decarboxylase was shown to be a monomer and is the first such enzyme to which a sequence has been assigned. The other two proteins, encoded by NRRL3_10507 and NRRL3_04788, were active in assays for muconate isomerase and 3-oxoadipate enol lactone hydrolase, respectively, from the catechol branch of the pathway. Both enzymes were found to be dimers. In comparison, the bacterial muconate isomerase is a decameric enzyme with smaller subunits, while bacterial 3-oxoadipate enol lactone hydrolase is also dimeric.
These data have resulted in the identification of molecular functions for four genes encoding key enzymes in aromatic degradation by A. niger.