This manuscript reports structure–function studies of Catechol 2,3-dioxygenase (C23O64), which is the second enzyme in the metabolic degradation pathway of 3-nitrotoluene by Diaphorobacter sp. strain DS2. The recombinant protein is a ring cleavage enzyme for 3-methylcatechol and 4-methylcatechol products formed after dioxygenation of the aromatic ring. Here we report the substrate-free, substrate-bound, and substrate-analog bound crystal structures of C23O64. The protein crystallizes in the P6(2)22 space-group. The structures were determined by molecular replacement and refined to resolutions of 2.4, 2.4, 2.2 Å, respectively. A comparison of the structures with related extradiol dioxygenases showed 22 conserved residues. A comparison of the active site pocket with catechol 2,3-dioxygenase (LapB) from Pseudomonas sp KL28 and homoprotocatechuate 2,3-dioxygenase (HPCD) from Brevibacterium fuscum shows significant similarities to suggest that the mechanism of enzyme action is similar to HPCD.
