Substrate Binding Mechanism of a Type I 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 AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a ␤-hairpin structure and a C-terminal tail. 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 revealed that the C-terminal tail and ␤-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.A general comparison of the major pathways for the aerobic degradation of aromatic hydrocarbons in bacteria has revealed that the initial conversion steps are carried out by different enzymes, but that the compounds are transformed into a limited number of central intermediates, including (substituted) catechols (1). These dihydroxylated intermediates are channeled into one of two possible pathways: an ortho-cleavage pathway or a meta-cleavage pathway (2). The former pathway is catalyzed by intradiol dioxygenases, which utilize a non-heme ferric iron to cleave the aromatic ring ortho to the hydroxyl substituents, while the latter pathway is catalyzed by extradiol dioxygenases, which utilize a non-heme ferrous iron or other divalent metal ion to cleave the aromatic ring meta to the hydroxyl substituents. Additionally, it is generally understood that extradiol dioxygenase is more versatile than its intradiol counterpart (3).Two decade-long research on the reaction mechanism of extradiol dioxygenases has revealed that the catalytic process occurs via a two-step mechanism: binding of catechol substrates and incorporation of the dioxygen atoms of molecular oxygen into the substrate (3-7). As depicted in Fig. 1, the iron atom in the active site is coordinated by the so-called 2-His-1-carboxylate facial triad motif (two histidines and one glutamate (or aspartate)), which occupies only one face of the coordination sphere. The opposite face is occupied by a displaceable water molecule and the hydroxyl oxygens of the catechol substrate. The binding of the substrate triggers the release of the water, thereby opening a coordination site for molecular oxygen, which is then apparently activated by an electron transferred from the substrate through the iron. Several conserved amino...

Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium

“…In contrast, C23O2 showed higher specific activity toward substituted catechols. As the substrate specificity was in connection with protein structure to some extent 57 , the distinct substrate metabolism feature of the two enzymes suggested the two C23Os had different enzyme structure and function. In addition, this substrate specificity test also suggested that the two C23Os in this study played a major role in the downstream pathway of phenanthrene degredation.…”

Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

“…strain DK17 (Kim et al, 2005) cleaved 3-methylcatechol at a higher rate than catechol. Cho et al (2010) have revealed that in substrate-bound methylcatechol 2,3-dioxygenase from strain DK17 3-methylcatechol interacts with the iron via a single hydroxyl group. The β-hairpin structure of the active C-domain of the enzyme forms part of the substrate binding pocket, responsible for the substrate specificity as well as its correct positioning in the active site.…”

Theoretical investigation on proton transfer mechanism of extradiol dioxygenase

“…On the one hand, experimental studies have proved that the protonation of alkylperoxo is very important for O-O bond cleavage. 19,20 It is generally accepted that the proton caused the protonation of alkylperoxo comes from the monoanionic catechol substrate, which has been conrmed by the available UV resonance Raman spectroscopic and electronic absorption spectroscopic data 13,[21][22][23] and the asymmetric binding X-ray crystal structure of the enzyme-substrate complex. 24,25 However, the protonation mechanism of alkylperoxo is still under debate, because no intermediate is captured by experimental methods before the formation of Fe(II)-alkyl(hydro) peroxo species in wild-type (WT) enzyme reaction.…”

“…[25][26][27] Considering the above two aspects, several researchers suggested that His200 could participate in the proton-transfer process and speculated that the monoanionic substrate gave a proton to the His200, which was then passed to superoxide anion. 17,19 In order to explain the above mechanism in more detail, attempts based on DFT calculation have been carried out. [28][29][30][31] Christian and coworkers 28 suggested that the proton-transfer and oxygen addition process consisted of several steps (Scheme 2): (a) His200 abstracted a proton from the hydroxyl group of the catecholic substrate.…”