Catechol 2,3-dioxygenase isoenzymes from Diaphorabacter sp. strain DS2 demonstrate different substrate preferences.

Two monomeric isozymes of catechol 2,3-dioxygenase C23O64 and C23O68 (EC 1.13.11.2) from Diaphorobacter sp. strain DS2 were cloned and overexpressed in E. coli BL21. The genes (C23O64 and C23O68) have open reading frames of 945 and 927 bp, respectively, with 85 % nucleotide and 42 % amino acid residue homology. These isozymes catalyze the m-cleavage of catechol ring to produce the corresponding 2-hydroxymuconic semialdehyde (2-HMS). C23O64 exhibited strong activity exclusively with 4-methyl catechol and 3-methyl catechol, but C23O68 preferred 4-chloro catechol. The isozymes showed maximal activity at 37 °C. Glutathione, mercaptoethanol, and DTT inhibited enzyme activity, while ascorbic acid and 1,10-phenanthroline enhanced enzymatic activity. The oxidizing agent HO inhibited the activity of both enzymes. The two enzymes are similar in molecular weight and catalytic parameters; however, their substrate specificity varies towards substituted catechols, thereby giving the organism a significant advantage when growing on a mixture of pollutants.