Substrate-Binding Mechanism of a TypeⅠ Extradiol Dioxygenase

Abstract

A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of Selenomethionine-substituted AkbC, a type I extradiol dioxygenase, and the enzyme-substrate (3-methylcatechol) complex revealed the substrate-binding process of extradiol dioxygenase. AkbC was found to be composed of eight identical subunits, each containing one ferrous ion. AkbC is composed of an N-domain (residues M1-G138) and an active C-domain (K139-G284), which contains iron coordinated by a 2-His-1-carboxylate facial triad motif (H149, H212, and E263). The C-domain includes a β-hairpin structure (β11-β12) and a C-terminal tail (H285-G300). In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate-binding process. Structure-based mutagenesis combined with structural analyses reveals that the C-terminal tail and β-hairpin form part of the substrate-binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate-binding mechanism is proposed.