Preparation of human drug metabolites using fungal peroxygenases

Poraj-Kobielska, Marzena; Kinne, Matthias; Ullrich, René; Scheibner, Katrin; Kayser, Gernot; Hammel, Kenneth E.; Hofrichter, Martin

doi:10.1016/j.bcp.2011.06.020

Cited by 69 publications

(67 citation statements)

References 63 publications

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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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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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281

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“…UPOs have successfully been used as oxygenation tools for the synthesis of human drug metabolites 17, 18, 19. The first detailed studies on steroid hydroxylation by several UPOs revealed preferred accessibility of the hydrophobic C17 alkyl side chain to the substrate channel; the entrance of whole rings was found to be energetically penalized, in particular when the C3 position was already oxyfunctionalized 17, 20. ‐21 Consequently, substrates, such as testosterone, that lack an alkyl side chain or bearing oxidized C3 were not converted 20…”

Section: Introductionmentioning

confidence: 99%

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

et al. 2017

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Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

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“…As such, they are proving to be unusually efficient biocatalysts (27). Numerous drug compounds are efficiently oxidized to products that are often the same as those from mammalian CYP transformations (28). These properties have also afforded a rare and revealing opportunity to study the reaction steps in C−H bond hydroxylation events with a hemethiolate protein that is not a CYP, but which may function in analogous ways.…”

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confidence: 99%

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

Wang

Ullrich

Hofrichter

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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A kinetic and spectroscopic characterization of the ferryl intermediate (APO-II) from APO, the heme-thiolate peroxygenase from Agrocybe aegerita, is described. APO-II was generated by reaction of the ferric enzyme with metachloroperoxybenzoic acid in the presence of nitroxyl radicals and detected with the use of rapidmixing stopped-flow UV-visible (UV-vis) spectroscopy. The nitroxyl radicals served as selective reductants of APO-I, reacting only slowly with APO-II. APO-II displayed a split Soret UV-vis spectrum (370 nm and 428 nm) characteristic of thiolate ligation. Rapid-mixing, pHjump spectrophotometry revealed a basic pK a of 10.0 for the Fe IV −O−H of APO-II, indicating that APO-II is protonated under typical turnover conditions. Kinetic characterization showed that APO-II is unusually reactive toward a panel of benzylic C−H and phenolic substrates, with second-order rate constants for C−H and O−H bond scission in the range of 10-10 7 M −1 ·s −1 . Our results demonstrate the important role of the axial cysteine ligand in increasing the proton affinity of the ferryl oxygen of APO intermediates, thus providing additional driving force for C−H and O−H bond scission.

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Preparation of human drug metabolites using fungal peroxygenases

Cited by 69 publications

References 63 publications

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

Directed Evolution of Unspecific Peroxygenase from Agrocybe aegerita

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

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