A substrate-binding-state mimic of H<sub>2</sub>O<sub>2</sub>-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates

Okada, Shoji; Fujishiro, Takashi; Nishio, K.; Kano, Yukiko; Kimoto, Hiroshi; Chien, Shih-Cheng; Onoda, Hiroki; Muramatsu, Akinori; Tanaka, Shota; Hori, Ayumi; Sugimoto, Hiroshi; Shiro, Yoshitsugu; Watanabe, Yoshihito

doi:10.1039/c6cy00630b

Cited by 56 publications

(88 citation statements)

References 52 publications

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“…In summary, we have constructed a unique approach for generating P450BM3 peroxygenase activity through the use of exogenous DFSMs. Our system achieved the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450‐H 2 O 2 system reported to date . We believe that this system can be further improved by optimizing the structure of the DFSMs in combination with rational design of the enzyme.…”

Section: Figurementioning

confidence: 82%

“…The enantiomeric excess of the ( R )‐enantiomer was further improved to 91 % for the combination of F87A‐T268V/Im‐C6‐Phe. To our knowledge, this represents the highest enantioselectivity of styrene epoxidation achieved by P450s . In addition, DFSMs with various acyl amino acid groups produced different effects on the change in enantioselectivity (Table , Figure S13).…”

Section: Figurementioning

confidence: 91%

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Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Chen

et al. 2018

Angewandte Chemie

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We report a unique strategy for the development of a H2O2‐dependent cytochrome P450BM3 system, which catalyzes the monooxygenation of non‐native substrates with the assistance of dual‐functional small molecules (DFSMs), such as N‐(ω‐imidazolyl fatty acyl)‐l‐amino acids. The acyl amino acid group of DFSM is responsible for bounding to enzyme as an anchoring group, while the imidazolyl group plays the role of general acid–base catalyst in the activation of H2O2. This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450–H2O2 system previously reported. This work provides the first example of the activation of the normally H2O2‐inert P450s through the introduction of an exogenous small molecule. This approach improves the potential use of P450s in organic synthesis as it avoids the expensive consumption of the reduced nicotinamide cofactor NAD(P)H and its dependent electron transport system. This introduces a promising approach for exploiting enzyme activity and function based on direct chemical intervention in the catalytic process.

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Section: Figurementioning

confidence: 82%

Section: Figurementioning

confidence: 91%

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Chen

et al. 2018

Angewandte Chemie

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“… 144 , 145 Recently, Watanabe et al found that the mutation of the highly conserved threonine (T268 in P450 BM3 , Figure 13 ) into glutamic acid substantially enhanced the peroxygenase activity of a number of P450s, including P450 BM3 , P450 cam , and CYP119. 146 The glutamic acid mutation is thought to mimic the role of the substrate carboxyl group in CYP152 for O–O bond activation.…”

Section: Key Intermediates Involved In O 2 Activatmentioning

confidence: 99%

“… 306 , 408 , 409 In several alkene oxidation reactions, a cationic intermediate, presumably generated via single electron transfer from alkyl radical to the ferryl center, was inferred from the formation of aldehydes, ketone, or heme N -alkylated side products. 17 , 92 , 146 , 410 , 411 The ketone or aldehyde products were generated via a 1,2-hydride shift, while heme N -alkylation products were likely to be formed via direct electrophilic attack of the carbocation from a pyrrole nitrogen of the porphyrin ligand. 412 Very recently, Hammer, Arnold, and co-workers have harnessed the side activity of P450s for aldehyde formation to develop the first olefin anti-Markovnikov oxygenase (aMOx) via directed evolution ( Figure 45 b).…”

Section: C–h Functionalization Reactions Mediated By Oxoiron(iv) Porpmentioning

confidence: 99%

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

2017

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As a result of the adaptation of life to an aerobic environment, nature has evolved a panoply of metalloproteins for oxidative metabolism and protection against reactive oxygen species. Despite the diverse structures and functions of these proteins, they share common mechanistic grounds. An open-shell transition metal like iron or copper is employed to interact with O2 and its derived intermediates such as hydrogen peroxide to afford a variety of metal–oxygen intermediates. These reactive intermediates, including metal-superoxo, -(hydro)peroxo, and high-valent metal–oxo species, are the basis for the various biological functions of O2-utilizing metalloproteins. Collectively, these processes are called oxygen activation. Much of our understanding of the reactivity of these reactive intermediates has come from the study of heme-containing proteins and related metalloporphyrin compounds. These studies not only have deepened our understanding of various functions of heme proteins, such as O2 storage and transport, degradation of reactive oxygen species, redox signaling, and biological oxygenation, etc., but also have driven the development of bioinorganic chemistry and biomimetic catalysis. In this review, we survey the range of O2 activation processes mediated by heme proteins and model compounds with a focus on recent progress in the characterization and reactivity of important iron–oxygen intermediates. Representative reactions initiated by these reactive intermediates as well as some context from prior decades will also be presented. We will discuss the fundamental mechanistic features of these transformations and delineate the underlying structural and electronic factors that contribute to the spectrum of reactivities that has been observed in nature as well as those that have been invented using these paradigms. Given the recent developments in biocatalysis for non-natural chemistries and the renaissance of radical chemistry in organic synthesis, we envision that new enzymatic and synthetic transformations will emerge based on the radical processes mediated by metalloproteins and their synthetic analogs.

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“…The ee value was further improved (to 91 %) with the combination of F87A/T268V and Im‐C6‐Phe. This result represents the highest enantioselectivity observed for styrene epoxidation by P450s . The potentially important role of DFSMs in contributing to the generation of chirality suggests that the DFSM‐facilitated P450BM3–H 2 O 2 system holds promise in the development of an enantioselective approach for P450‐catalyzed styrene epoxidation.…”

Section: Dual‐functional Small Molecule Co‐catalysis (Dfsm)mentioning

confidence: 99%

Strategies for Substrate‐Regulated P450 Catalysis: From Substrate Engineering to Co‐catalysis

Wang

Cong

2019

Chemistry A European J

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Cytochrome P450 enzymes (P450s) catalyze the monooxygenation of various organic substrates. These enzymes are fascinating and promising biocatalysts for synthetic applications. Despite the impressive abilities of P450s in the oxidation of C−H bonds, their practical applications are restricted by intrinsic drawbacks, such as poor stability, low turnover rates, the need for expensive cofactors (e.g., NAD(P)H), and the narrow scope of useful non‐native substrates. These issues may be overcome through the general strategy of protein engineering, which focuses on the improvement of the catalysts themselves. Alternatively, several emerging strategies have been developed that regulate the P450 catalytic process from the viewpoint of the substrate. These strategies include substrate engineering, decoy molecule, and dual‐functional small‐molecule co‐catalysis. Substrate engineering focuses on improving the substrate acceptance and reaction selectivity by means of an anchoring group. The latter two strategies utilize co‐substrate‐like small molecules that either are proposed to reform the active site, thereby switching the substrate specificity, or directly participate in the catalytic process, thereby creating new catalytic peroxygenation capabilities towards non‐native substrates. For at least 10 years, these approaches have played unique roles in solving the problems highlighted above, either alone or in conjunction with protein engineering. Herein, we review three strategies for substrate regulation in the P450‐catalyzed oxidation of non‐native substrates. Furthermore, we address remaining challenges and potential solutions associated with these approaches.

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A substrate-binding-state mimic of H₂O₂-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates

Abstract: H2O2-dependent cytochrome P450s that can catalyze monooxygenation of nonnative substrates were constructed by one-point mutagenesis.

Cited by 56 publications

References 52 publications

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

Strategies for Substrate‐Regulated P450 Catalysis: From Substrate Engineering to Co‐catalysis

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A substrate-binding-state mimic of H2O2-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates

Abstract: H2O2-dependent cytochrome P450s that can catalyze monooxygenation of nonnative substrates were constructed by one-point mutagenesis.

Cited by 56 publications

References 52 publications

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Dual‐Functional Small Molecules for Generating an Efficient Cytochrome P450BM3 Peroxygenase

Oxygen Activation and Radical Transformations in Heme Proteins and Metalloporphyrins

Strategies for Substrate‐Regulated P450 Catalysis: From Substrate Engineering to Co‐catalysis

Contact Info

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A substrate-binding-state mimic of H₂O₂-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates