Conversion of extradiol aromatic ring-cleaving homoprotocatechuate 2,3-dioxygenase into an intradiol cleaving enzyme

The intra- and extradiol subfamilies of catechol-adduct ring-cleaving dioxygenases each exhibit nearly absolute fidelity for the ring cleavage position. This is often attributed to the fact that the oxygen activation mechanism of intradiol dioxygenases utilizes Fe³⁺ while that of the extradiol enzymes employs Fe²⁺, but the subfamilies also differ in primary sequence, structural fold, iron ligands, and second sphere active site amino acid residues. Here, we examine the effects of the second sphere residue H200 in the active site of homoprotocatechuate 2,3-dioxygenase (2,3-HPCD), an extradiol-cleaving enzyme. It is shown that the H200F mutant enzyme catalyzes extradiol cleavage of the normal substrate, homoprotocatechuate (HPCA), but intradiol cleavage of the alternative substrate 2,3-dihydroxybenzoate (2,3-DHB) while in the Fe²⁺ oxidation state. Wild-type 2,3-HPCD catalyzes extradiol cleavage of both substrates. This is the first report of intradiol cleavage by an extradiol dioxygenase. It suggests that intradiol cleavage can occur with the iron in the Fe²⁺ state, with the iron ligand set characteristic of extradiol dioxygenases, and through a mechanism in which oxygen is activated by binding to the iron rather than directly attacking the substrate as in true intradiol dioxygenases. This indicates that substrate binding geometry and acid/base chemistry of second sphere residues play important roles in determining the course of the dioxygenase reaction.