Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita

Kluge, Martin; Ullrich, René; Scheibner, Katrin; Hofrichter, Martin

doi:10.1039/c1gc16173c

Cited by 114 publications

(90 citation statements)

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“…The most detailedly studied member of this group is the Agrocybe aegerita aromatic peroxygenase ( Aae APO), which is secreted by an agaric fungus, the Black poplar mushroom. Aae APO was found to catalyze a variety of oxyfunctionalization reactions such as hydroxylation of saturated hydrocarbons (Kluge et al 2012; Peter et al 2011; Ullrich and Hofrichter 2005), epoxidation of unsaturated hydrocarbons (Kluge et al 2009; Kluge et al 2012), heterocyclic N -oxidation (Ullrich et al 2008), sulfoxidation (Aranda et al 2009) and O -dealkylation (ether cleavages (Kinne et al 2009b)). In contrast to the distantly related chloroperoxidase from Caldariomyces fumago (CPO; EC 1.11.1.10), APOs are able to peroxygenate/hydroxylate aromatic hydrocarbons, e.g.…”

Section: Introductionmentioning

confidence: 99%

Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita

et al. 2013

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Aromatic peroxygenase (APO) is an extracellular enzyme produced by the agaric basidiomycete Agrocybe aegerita that catalyzes diverse peroxide-dependent oxyfunctionalization reactions. Here we describe the oxygenation of the unactivated aromatic ring of benzene with hydrogen peroxide as co-substrate. The optimum pH of the reaction was around 7 and it proceeded via an initial epoxide intermediate that re-aromatized in aqueous solution to form phenol. Identity of the epoxide intermediate as benzene oxide was proved by a freshly prepared authentic standard using GC-MS and LC-MS analyses. Second and third [per]oxygenation was also observed and resulted in the formation of further hydroxylation and following [per]oxidation products: hydroquinone and p-benzoquinone, catechol and o-benzoquinone as well as 1,2,4-trihydroxybenzene and hydroxy-p-benzoquinone, respectively. Using H218O2 as co-substrate and ascorbic acid as radical scavenger, inhibiting the formation of peroxidation products (e.g., p-benzoquinone), the origin of the oxygen atom incorporated into benzene or phenol was proved to be the peroxide. Apparent enzyme kinetic constants (kcat, Km) for the peroxygenation of benzene were estimated to be around 8 s-1 and 3.6 mM. These results raise the possibility that peroxygenases may be useful for enzymatic syntheses of hydroxylated benzene derivatives under mild conditions.

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Section: Introductionmentioning

confidence: 99%

Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita

et al. 2013

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“…Since its discovery 10 years ago, the potential use of UPO in applications ranging from chemical processes (including some relevant industrial transformations, such as alkane hydroxylation and olefin epoxidation) to the preparation of O-and N-dealkylated human drug metabolites, as well as bioremediation (polycyclic aromatic hydrocarbon [PAH] oxidation) and biosensor development, has been studied exhaustively (41)(42)(43)(44)(45)(46)(47)(48)(49)(50). For decades, regio-and enantioselective oxyfunctionalization has been a "forbidden territory" for most biocatalysts, except for P450 The stabilities of wt UPO1 (C) and the PaDa-I mutant (D) after incubation for 48 h in 50% organic cosolvents were assessed by incubating enzyme samples in 100 mM potassium phosphate buffer (pH 7.0) containing 50% (vol/vol) organic cosolvent in screw-cap vials.…”

Section: Y A[14]v R[15]g A[21]d Mutations)mentioning

confidence: 99%

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Molina‐Espeja

García-Ruiz

González-Pérez

et al. 2014

Appl Environ Microbiol

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c Unspecific peroxygenase (UPO) represents a new type of heme-thiolate enzyme with self-sufficient mono(per)oxygenase activity and many potential applications in organic synthesis. With a view to taking advantage of these properties, we subjected the Agrocybe aegerita UPO1-encoding gene to directed evolution in Saccharomyces cerevisiae. To promote functional expression, several different signal peptides were fused to the mature protein, and the resulting products were tested. Over 9,000 clones were screened using an ad hoc dual-colorimetric assay that assessed both peroxidative and oxygen transfer activities. After 5 generations of directed evolution combined with hybrid approaches, 9 mutations were introduced that resulted in a 3,250-fold total activity improvement with no alteration in protein stability. A breakdown between secretion and catalytic activity was performed by replacing the native signal peptide of the original parental type with that of the evolved mutant; the evolved leader increased functional expression 27-fold, whereas an 18-fold improvement in the k cat /K m value for oxygen transfer activity was obtained. The evolved UPO1 was active and highly stable in the presence of organic cosolvents. Mutations in the hydrophobic core of the signal peptide contributed to enhance functional expression up to 8 mg/liter, while catalytic efficiencies for peroxidative and oxygen transfer reactions were increased by several mutations in the vicinity of the heme access channel. Overall, the directed-evolution platform described is a valuable point of departure for the development of customized UPOs with improved features and for the study of structure-function relationships.

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“…10a,18 Thus, slow addition of 20 mM H 2 O 2 or peroxyacid to a solution containing 0.22 µM Aae APO and 10 mM p –ethylbenzoic acid produced ( R )-4-(1-hydroxyethyl)benzoic acid in high conversion and 99% ee (Figure S2). 17 Subsequent oxidation of the benzylic alcohol product occurred only after all of the p –ethylbenzoic acid had been consumed. Remarkably, Aae APO was found to hydroxylate a methyl C–H bond in dimethyl butyric acid and even neopentane (BDE ~ 100 kcal/mol) to produce the corresponding primary alcohols.…”

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Detection and Kinetic Characterization of a Highly Reactive Heme–Thiolate Peroxygenase Compound I

et al. 2012

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The extracellular heme-thiolate peroxygenase from Agrocybe aegerita (AaeAPO) has been shown to hydroxylate alkanes and numerous other substrates using hydrogen peroxide as the terminal oxidant. We describe the kinetics of formation and decomposition of AaeAPO compound I upon its reaction with mCPBA. The UV–vis spectral features of AaeAPO–I (361, 694 nm) are similar to those of chloroperoxidase–I and the recently–described cytochrome P450–I. The second–order rate constant for AaeAPO–I formation was 1.0 (±0.4) ×107 M−1s−1 at pH 5.0, 4 °C. The relatively slow decomposition rate, 1.4 (±0.03) s−1, allowed the measurement of its reactivity toward a panel of substrates. The observed rate constants, k2’, spanned five orders of magnitude and correlated linearly with bond dissociation enthalpies of strong C–H bond substrates with a log k2’ vs. BDE slope of ~ 0.4. However, the hydroxylation rate was insensitive to C–H BDE below 90 kcal/mol, similar to the behavior of the t-butoxy radical. The shape and slope of the Brønsted-Evans-Polanyi plot indicate a symmetrical transition state for the stronger C–H bonds and suggest entropy control of the rate in an early transition state for weaker C–H bonds. The AaeAPO–II FeIVO–H BDE was estimated to be ~ 103 kcal/mol. All results support the formation of a highly reactive AaeAPO oxoiron(IV) porphyrin radical cation intermediate that is the active oxygen species in these hydroxylation reactions.

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Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita

Cited by 114 publications

References 59 publications

Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita

Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Detection and Kinetic Characterization of a Highly Reactive Heme–Thiolate Peroxygenase Compound I

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