Efficient biocatalytic C–H bond oxidation: an engineered heme-thiolate peroxygenase from a thermostable cytochrome P450

Gee, Alecia R.; Stone, Isobella S. J.; Stockdale, Tegan P.; Pukala, Tara L.; De Voss, James J.; Bell, Stephen G.

doi:10.1039/d3cc04626e

Cited by 4 publications

(4 citation statements)

References 26 publications

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“…Deploying the I-helix of the CYP255 family may enable the retrofitting of peroxygenase activity into other P450s, making biocatalysis more favorable for larger scale applications. Indeed, the peroxygenase activity of CYP119 (46) and CYP199, CYP154C8 and CYP102 (47) was improved by mutating the acid-alcohol pair and surrounding residues to resemble the I-helix of the CYP255 enzymes.…”

Section: Discussionmentioning

confidence: 99%

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Harlington,

Das,

Shearwin

et al. 2024

Preprint

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TheO-demethylation of lignin aromatics is a rate-limiting step in their bioconversion to high-value compounds. A recently discovered cytochrome P450 enzyme SyoA was found to demethylate the sinapyl alcohol-derived (S-lignin) aromatic syringol. In this work, we solved high-resolution X-ray crystal structures of SyoA in the substrate-free and substrate-bound states and evaluate the demethylation ofpara-substituted S-lignin aromatics via the monooxygenase pathway and peroxide shunt pathway. We found that SyoA demethylates S-lignin aromatics with the following activity: 4-methylsyringol > syringaldehyde > syringol exclusively using the peroxide shunt pathway. The atomic-resolution structure of SyoA reveals the position of the non-canonical residues in the I-helix (Gln252 and Glu253). Site-directed mutagenesis of this amide-acid pair of a homologous CYP255 enzyme GcoA, which can catalyze the O-demethylation of guaiacol using both monooxygenase and peroxygenase activity, showed the amide-acid pair is critical for both pathways. This work expands the enzymatic toolkit for improving the capacity to funnel lignin towards high-value compounds, and defines the new chemistry within the active site of the enzyme that enables efficient peroxygenase activity. These insights provide a framework for engineered peroxygenase activity in other cytochrome P450 enzymes, with the potential for more facile catalysis, relative to traditional P450 monooxygenases which require difficult to handle redox partners and expensive nicotinamide cofactors.

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Section: Discussionmentioning

confidence: 99%

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Harlington,

Das,

Shearwin

et al. 2024

Preprint

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“…This would move the glutamate into the position observed in GcoA, which may result in more efficient catalysis. This has been demonstrated to work in the hyperthermophilic CYP119A1 enzyme of Sulfolobus acidocalderus, by replacing a seven amino acid section of the I-helix residues with those found in the CYP255 family enzymes …”

Section: Introductionmentioning

confidence: 99%

“…This has been demonstrated to work in the hyperthermophilic CYP119A1 enzyme of Sulfolobus acidocalderus, by replacing a seven amino acid section of the I-helix residues with those found in the CYP255 family enzymes. 52 Here, we alter the residues which constitute the oxygenbinding groove of CYP199A4 ( 248 AGLDTTV 254 ) so that it more closely resembles the sequence of GcoA ( 245 GAMQEPG 251 ), to investigate whether this leads to enhanced peroxygenase activity. We propose that the double mutant, which replaces D251 with Q and T252 with E in CYP199A4, would improve peroxygenase activity.…”

Section: ■ Introductionmentioning

confidence: 99%

“…We have previously demonstrated that we could improve the peroxygenase activity of the hyperthemophilic CYP119 enzyme from an archaeum by replacing the seven amino acids of its oxygen-binding groove that align with those investigated here. 52 Given the lack of soluble folded enzyme when we replaced the equivalent seven amino acids of CYP199A4 and the improvements observed in the CYP199A4 enzyme with the QE double mutant, we wished to assess if this strategy would work with other cytochrome P450 monooxygenase enzymes. We chose the bacterial species CYP154C8 which can regio-and stereoselectively oxidize progesterone to 16α-hydroxyprogesterone.…”

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confidence: 99%

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Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region

Podgorski,

Akter,

Churchman

et al. 2024

ACS Catal.

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Cytochrome P450 enzymes (CYPs) are biocatalysts for the generation of fine chemicals including natural products, drug metabolites, and flavor and fragrance compounds. However, both the high cost of the required nicotinamide cofactors and their need for additional electron transfer proteins limit their use. Here, we investigate whether CYPs can be converted into more efficient peroxygenases through protein engineering of the enzyme's oxygen activation machinery. We improve the peroxygenase activity by modifying selected residues within the I-helix to more closely resemble those of a natural peroxygenase. We produced mutants containing two, four, and six mutations, within this region of the Ihelix. In our model CYP system, the double mutant in which glutamine and glutamate residues replaced aspartate and threonine, respectively, was found to have significantly higher peroxygenase activity for the O-demethylation of 4-methoxybenzoic acid than a single glutamate mutant prototype. Importantly, it functioned better at lower H 2 O 2 concentrations and could convert all the added substrate to product. All the mutants maintained the stereoselectivity of the CYP enzyme for the epoxidation of 4-vinylbenzoic acid. The X-ray crystal structures of these enzymes showed significant structural changes at the oxygen-binding groove in the I-helix. In crystallo reactions with 4-methylbenzoic acid exhibit electron density corresponding to the 4-(hydroxymethyl)benzoic acid metabolite. We extended this mutagenesis strategy to a bacterial steroid-hydroxylating CYP and an uncharacterized CYP from a thermophilic bacterium. In these instances, we generate peroxygenases, which catalyze the regio-and stereoselective hydroxylation of progesterone and the hydroxylation of fatty acids at low hydrogen peroxide concentrations.

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Ultrathin 2D Porphyrin‐Based Zr‐MOF Nanosheets as Heterogeneous Catalysts for Styrene Epoxidation and Benzylic C‐H Oxidation

Wang,

Ren,

Feng

et al. 2024

ChemCatChem

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Selective oxidation of hydrocarbons using molecular oxygen as sole oxidant under mild conditions remains a challenging task. In this context, metal‐organic frameworks (MOFs) have been widely used in various oxidation reactions due to their porosity, high surface area and designability. However, the slow diffusion of substrates/products in micropores of three‐dimensional (3D) bulk MOFs hinders the efficient catalytic performance of such materials. Herein an ultrathin two‐dimensional (2D) porphyrin‐based Zr‐MOF nanosheet (Zr‐TCPP) is synthesized through modulator‐control strategy. Subsequently, various metal ions are anchored into the porphyrin ring by post synthesis modification to afford a series of 2D Zr‐TCPP(M) (M=Mn, Fe, Co, Ni, Cu and Zn). Various structural characterization techniques indicate Zr‐TCPP(M) is nanoflower structure with ultrathin nanoplate petals which provides fully exposed accessible active sites. Among them, Zr‐TCPP(Fe) shows excellent catalytic performance in styrene epoxidation reactions and benzylic C‐H oxidation reactions using O2 as sole oxidant under ambient temperature and pressure. The remarkable activity arises from high density of exposed porphyrin‐Fe active sites, low diffusion barriers for substrates and products, as well as a similar homogeneous reaction space. Furthermore, Zr‐TCPP(Fe) nanosheet is easily recycled by centrifugation and reused at least five times without significant loss of catalytic activity.

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Efficient biocatalytic C–H bond oxidation: an engineered heme-thiolate peroxygenase from a thermostable cytochrome P450

Abstract: A highly sought after reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds. We demonstrate the hydroxylation of fatty acids using an engineered thermostable archaeal cytochrome P450 enzyme....

Cited by 4 publications

References 26 publications

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

The S-ligninO-demethylase SyoA: Structural insights into a new class of heme peroxygenase enzymes

Engineering Peroxygenase Activity into Cytochrome P450 Monooxygenases through Modification of the Oxygen Binding Region

Ultrathin 2D Porphyrin‐Based Zr‐MOF Nanosheets as Heterogeneous Catalysts for Styrene Epoxidation and Benzylic C‐H Oxidation

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