2,3-Cleavage of Substituted Catechols in Nocardia sp. DSM 43251 (Rhodococcus rubrus)

A catechol 2.3-dioxygenase, isolated from Nocardia sp. DSM 43251 (Rhodococcus rubrus) after induction with unpolar substituted phenols e.g. 4-(methylthio)-phenol or 4-methoxyphenol, was purified 79-fold by ammonium sulfate precipitation, acetone precipitation, and sephadex G 200 gel filtration. The molecular weight, as determined by gel filtration, was 125,000. SDS-polyacrylamide gel electrophoresis revealed three non-identical subunits with molecular weights from 40,000 to 50,000. Highest activity was obtained at pH 7.5 to 8.5. The enzyme was stable in the presence of oxygen, reducing and oxidizing agents, and at high temperatures with an optimum temperature of 60 °C. Atomic absorption spectrometry proved one atom of iron per molecule of enzyme which could not be removed by dialysis. The ring cleavage reaction was inhibited by metal ions like Ag⁺, Hg⁺⁺ and Cu⁺⁺ (10⁻⁶–10⁻⁵ M), and by 2,2′-bipyridyl (10⁻³ M).

Catechol 2,3-dioxygenase from Nocardia sp. DSM 43251 showed a strict specificity for catechols with a substituent at C-4. Maximum reaction velocity was strongly influenced by the electronegativity of these substituents. Additional methyl groups at C-3 or C-5 decreased the affinity of the enzyme for the substrate as well as the reaction velocity. The structure of the reaction product and linear correlation of the reaction velocity with the tfp-values of the different catechols tested clearly indicate a 2,3-cleavage-mechanism.