Enabling highly (<i>R</i>)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases

Zhao, Panxia; Chen, Jie; Ma, Ning; Chen, Jingfei; Qin, Xiangquan; Liu, Chuanfei; Yao, Fuquan; Yao, Lishan; Jin, Longyi; Cong, Zhiqi

doi:10.1039/d1sc00317h

Cited by 55 publications

(40 citation statements)

References 53 publications

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“…This approach also gave modest to very good TONs (362-3480) and high (R)-enantioselectivities (95-99% ee) for the epoxidation of various styrene derivatives (Figure 5C), being comparable with the best (R)-enantioselective styrene monooxygenases, such as SeStyA from Streptomyces exfoliatus, AaStyA from Amycolatopsis albispora, and PbStyA from Pseudonocardiaceae reported recently [109,110]. The further semi-preparative scale experiments suggest its potential application in styrene epoxidation [92].…”

Section: Catalytic Applications Of the Dfsm-facilitated P450 Peroxyge...supporting

confidence: 56%

“…Therefore, developing the catalytic potential of the artificial P450 peroxygenase is not only expected to expand the chemical space of P450 enzymes but also act as a beneficial supplement to the relatively scarce natural peroxygenase resources in nature. In fact, the DFSM-facilitated P450BM3-H 2 O 2 system has shown versatile unique catalytic activity towards the peroxygenation reaction of various non-native substrates, such as epoxidation, hydroxylation, and sulfoxidation [66,[92][93][94][95].…”

Section: Catalytic Applications Of the Dfsm-facilitated P450 Peroxyge...mentioning

confidence: 99%

“…However, research interest has focused on the monooxygenase and peroxygenase activities of P450s, and only a handful of studies have examined the catalytic peroxidase functionality of P450s. The non-natural DFSM-facilitated P450-H 2 O 2 system described above mainly catalyzes various per-oxygenation reactions, including epoxidation, hydroxylation, and sulfoxidation [66,[92][93][94][95]. Interestingly, the oxidation of guaiacol, a classical substrate of peroxidases [141][142][143][144], catalyzed by the DFSM-facilitated P450BM3-H 2 O 2 system yielded demethylated catechol as a major product, suggesting it mainly functioned as a peroxygenase but not as a peroxidase [94].…”

Section: Switching Peroxidase Activity Of the Dfsm-facilitated P450 P...mentioning

confidence: 99%

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A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

Fan

Jiang

et al. 2022

Antioxidants

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Cytochrome P450 monooxygenases (P450s) are promising versatile oxidative biocatalysts. However, the practical use of P450s in vitro is limited by their dependence on the co-enzyme NAD(P)H and the complex electron transport system. Using H2O2 simplifies the catalytic cycle of P450s; however, most P450s are inactive in the presence of H2O2. By mimicking the molecular structure and catalytic mechanism of natural peroxygenases and peroxidases, an artificial P450 peroxygenase system has been designed with the assistance of a dual-functional small molecule (DFSM). DFSMs, such as N-(ω-imidazolyl fatty acyl)-l-amino acids, use an acyl amino acid as an anchoring group to bind the enzyme, and the imidazolyl group at the other end functions as a general acid-base catalyst in the activation of H2O2. In combination with protein engineering, the DFSM-facilitated P450 peroxygenase system has been used in various oxidation reactions of non-native substrates, such as alkene epoxidation, thioanisole sulfoxidation, and alkanes and aromatic hydroxylation, which showed unique activities and selectivity. Moreover, the DFSM-facilitated P450 peroxygenase system can switch to the peroxidase mode by mechanism-guided protein engineering. In this short review, the design, mechanism, evolution, application, and perspective of these novel non-natural P450 peroxygenases for the oxidation of non-native substrates are discussed.

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Section: Catalytic Applications Of the Dfsm-facilitated P450 Peroxyge...supporting

confidence: 56%

Section: Catalytic Applications Of the Dfsm-facilitated P450 Peroxyge...mentioning

confidence: 99%

Section: Switching Peroxidase Activity Of the Dfsm-facilitated P450 P...mentioning

confidence: 99%

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A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

Fan

Jiang

et al. 2022

Antioxidants

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“…Impressively, combined with enzyme mutagenesis, the DFSM based artificial P450BM3 peroxygenase has been successfully applied to oxidation of small alkenes which are with inert C−H bonds showing comparable turnover number to the H 2 O 2 ‐dependant natural alkane hydrolase [76] . In more recent studies, the effect of DFSM ( i. e ., Im‐C6‐Phe) on P450BM3 enzyme activity and enantioselectivities to synthesize ( R )‐styrene oxide has been further optimized by combination with enzyme engineering [78] . With the DFSM strategy, different P450BM3 variants displayed high O ‐demethylation on aromatic ethers [79] and peroxidase activity on guaiacol, 2,6‐dimethoxyphenol, o ‐phenylenediamine, and p ‐phenylenediamine [80] .…”

Section: Small Molecule Auxiliaries To Control P450s’ Activity For Sy...mentioning

confidence: 99%

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Zhang

Wang

2021

ChemBioChem

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Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy‐based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.

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“…Highly (R)-enantioselective epoxidation of styrenes has been achieved through protein engineering of a P450BM3 peroxygenase. 30 In combination with a dual-functional small molecule, N-(u-imidazolyl)-hexanoyl-L-phenylalanine (Im-C 6 -Phe), epoxidation of a range of o-, m-and p-halogenated styrenes was achieved with excellent enantioselectivities (Scheme 4).…”

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confidence: 99%

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Hill

Sutherland

2021

Nat. Prod. Rep.

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Enabling highly (R)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases

Abstract: Unlike the excellent (S)-enantioselective epoxidation of styrene performed by natural styrene monooxygenase (ee >99%), the (R)-enantioselective epoxidation of styrene has not yet achieved a comparable efficiency using natural or engineered...

Cited by 55 publications

References 53 publications

A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

A Unique P450 Peroxygenase System Facilitated by a Dual-Functional Small Molecule: Concept, Application, and Perspective

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

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