Investigation of acid–base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

Brivio, Michela; Schlosrich, Janne; Ahmad, Masroor; Tolond, Caroline; Bugg, Timothy D. H.

doi:10.1039/b821559f

Cited by 11 publications

(8 citation statements)

References 19 publications

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“…Such a transition results in the displacement of Tyr-253 and a water molecule, which leads to the deprotonation of the hydroxyl group of 3-MC, as well as the provision of a position for an oxygen molecule. This proposed process is in a good agreement with previous reports that the oxygen interaction occurs after the binding of the catechol substrate during the catalytic process of extradiol dioxygenases (3)(4)(5)(6)(7). In summary, this work shows that ring cleavage by the DK17 AkbC enzyme is preceded by a series of substrate-positioning steps, such that the flexible motifs direct and correctly position the substrate into the binding pocket/active site, allowing efficient substrate catalysis and product release.…”

Section: Discussionsupporting

confidence: 81%

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

Cho

Kim

Sohn

et al. 2010

Journal of Biological Chemistry

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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...

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Section: Discussionsupporting

confidence: 81%

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

Cho

Kim

Sohn

et al. 2010

Journal of Biological Chemistry

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“…We rule out the former which gives aN and aHβ values larger than those of the latter 25,26 . Since the other oxygen-centered radicals were not observed experimentally, these two radicals were probably not released from the intra-(at C1-C2 site) or extra-diol (at C1-C6 site) cleavages which was similar to those in catalysis by catechol dioxygenase or in chemical oxidation 20,21,[27][28][29][30][31][32] . Therefore, the trapped non-aromatic carbon-centered radical was assigned to the isomer of the o-benzosemiquinone radical, which was also detected in HPLC-MS ( Fig.2e).…”

Section: Resultsmentioning

confidence: 93%

“…Obviously, the formed obenzosemiquinone radical alone does not yield any adduct product. When adding laccase, a chelate complex was formed between the o-benzosemiquinone radical and the counterion Cu 1+ / Cu 2+ (T1) [19][20][21][27][28][29][30][31][32] . The inductive effect caused by Cu ion activates the cation radical tendency at the C4-C5 site.…”

Section: Resultsmentioning

confidence: 99%

Radical Mechanism of Laccase-Catalyzed Catechol Ring-Opening

Chen¹,

中国科学技术大学近代物理系²,

Wang³

et al. 2017

Acta Physico-Chimica Sinica

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Enzyme-catalyzed reactions are a prominent field of research in green chemistry. Laccase is a multicopper oxidase, which we used to study the oxidation of catechol. A mechanism for this ring-opening reaction is also proposed. A o-benzosemiquinone radical was the initial nascent product of catechol oxidation during the catalytic reaction. This radical underwent two reaction pathways: (1) formation of an intramolecular adduct, which gave a carbon-centered furan-derived radical trapped by 5,5-dimethyl-1-pyrroline-N-oxide (DMPO); (2) formation of an intermolecular adduct producing dimeric and trimeric oligomers, as resolved by mass spectrometry. Products of the furan-like intermediate were also characterized by 1 H-NMR. Simultaneously, a hydroxyl radical (•OH) originating from the water solvent was identified by 17 O-isotope tracing. The kinetics of this radical were also evident with substrates including 3-and 4-methyl catechol, but not with resorcinol and hydroquinone isomers, 3-and 4-nitro catechol, and 2,3-dihydroxynaphthalene. The mechanism of selective 620 CHEN Ming et al.: Radical Mechanism of Laccase-Catalyzed Catechol Ring-Opening No.3

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“…The pH was found to influence the activity of catechol 1,2-dioxygenase. Brivio et al [26] found that catechol 1,2-dioxygenase activity is independent of pH, in the pH range of 5.2–8.6. Silva et al [27] reported that catechol 1,2-dioxygenase activity was very low when pH value equaled to 4.…”

Section: Resultsmentioning

confidence: 99%

Aerobic Degradation of Trichloroethylene by Co-Metabolism Using Phenol and Gasoline as Growth Substrates

Wang

et al. 2014

IJMS

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Trichloroethylene (TCE) is a common groundwater contaminant of toxic and carcinogenic concern. Aerobic co-metabolic processes are the predominant pathways for TCE complete degradation. In this study, Pseudomonas fluorescens was studied as the active microorganism to degrade TCE under aerobic condition by co-metabolic degradation using phenol and gasoline as growth substrates. Operating conditions influencing TCE degradation efficiency were optimized. TCE co-metabolic degradation rate reached the maximum of 80% under the optimized conditions of degradation time of 3 days, initial OD600 of microorganism culture of 0.14 (1.26 × 107 cell/mL), initial phenol concentration of 100 mg/L, initial TCE concentration of 0.1 mg/L, pH of 6.0, and salinity of 0.1%. The modified transformation capacity and transformation yield were 20 μg (TCE)/mg (biomass) and 5.1 μg (TCE)/mg (phenol), respectively. Addition of nutrient broth promoted TCE degradation with phenol as growth substrate. It was revealed that catechol 1,2-dioxygenase played an important role in TCE co-metabolism. The dechlorination of TCE was complete, and less chlorinated products were not detected at the end of the experiment. TCE could also be co-metabolized in the presence of gasoline; however, the degradation rate was not high (28%). When phenol was introduced into the system of TCE and gasoline, TCE and gasoline could be removed at substantial rates (up to 59% and 69%, respectively). This study provides a promising approach for the removal of combined pollution of TCE and gasoline.

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Investigation of acid–base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

Cited by 11 publications

References 19 publications

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

Substrate Binding Mechanism of a Type I Extradiol Dioxygenase

Radical Mechanism of Laccase-Catalyzed Catechol Ring-Opening

Aerobic Degradation of Trichloroethylene by Co-Metabolism Using Phenol and Gasoline as Growth Substrates

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