Purification and properties of 2,3-dihydroxy-p-cumate-3,4-dioxygenase from Bacillus species.

2,3-Dihydroxy-p-cumate-3,4-dioxygenase, an enzyme involved in the catabolism of p-cymene, was purified to homogeneity from Bacillus species by affinity chromatography. Purification of the dioxygenase allowed the observation of the immediate ring cleavage product of 2,3-dihydroxy-p-cumate. The enzyme was optimally active at pH 8.2 and at 35 degrees C. The Km value for 2,3-dihydroxy-p-cumate was 32 microM. The enzyme had a broad substrate specificity for 3-substituted catechols. The activity of the enzyme was inhibited by heavy metals, sulphydryl inhibitors, iron-chelating agents, and substrate analogues. Fe2+ was suggested as a cofactor.