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Cytochrome P450 enzymes (CYPs) have attracted much promise as biocatalysts in a push for cleaner and more environmentally friendly catalytic systems. However, changing the substrate specificity of CYPs, such as CYP102A1, can be a challenging task, requiring laborious mutagenesis. An alternative approach is the use of decoy molecules that "trick" the enzyme into becoming active by impersonating the native substrate. Whilst the decoy molecule system has been extensively developed for CYP102A1, its general applicability for other CYP102-family enzymes has yet to be shown. Herein, we demonstrate that decoy molecules can "trick" CYP102A5 and A7 into becoming active and hydroxylating non-native substrates. Furthermore, significant differences in decoy molecule selectivity as well as decoy molecule binding were observed. The X-ray crystal structure of the CYP102A5 haem domain was solved at 2.8 Å, delivering insight into a potential substate-binding site that differs significantly from CYP102A1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Catching the structure of cytochrome P450 enzymes in flagrante is crucial for the development of P450 biocatalysts, as most structures collected are found trapped in a precatalytic conformation. At the heart of P450 catalysis lies Cpd I, a short-lived, highly reactive intermediate, whose recalcitrant nature has thwarted most attempts at capturing catalytically relevant poses of P450s. We report the crystal structure of P450BM3 mimicking the state in the precise moment preceding epoxidation, which is in perfect agreement with the experimentally observed stereoselectivity. This structure was attained by incorporation of the stable Cpd I mimic oxomolybdenum mesoporphyrin IX into P450BM3 in the presence of styrene. The orientation of styrene to the Mo-oxo species in the crystal structures sheds light onto the dynamics involved in the rotation of styrene to present its vinyl group to Cpd I. This method serves as a powerful tool for predicting and modelling the stereoselectivity of P450 reactions., (© 2022 Wiley-VCH GmbH.)

With the increasing demand for blue dyes, it is of vital importance to develop a green and efficient biocatalyst to produce indigo. This study constructed a hydrogen peroxide-dependent catalytic system for the direct conversion of indole to indigo using P450BM3 with the assistance of dual-functional small molecules (DFSM). The arrangements of amino acids at 78, 87, and 268 positions influenced the catalytic activity. F87G/T268V mutant gave the highest catalytic activity with k cat of 1402 min -1 and with a yield of 73%. F87A/T268V mutant was found to produce the indigo product with chemoselectivity as high as 80%. Moreover, F87G/T268A mutant was found to efficiently catalyze indole oxidation with higher activity ( k cat /K m = 1388 mM -1 min -1 ) than other enzymes, such as the NADPH-dependent P450BM3 (2.4-fold), the Ngb (32-fold) and the Mb (117-fold). Computer simulation results indicate that the arrangements of amino acid residues in the active site can significantly affect the catalytic activity of the protein. The DFSM-facilitated P450BM3 peroxygenase system provides an alternative, simple approach for a key step in the bioproduction of indigo.

We report an OmpF loop deletion mutant, which improves the cellular uptake of external additives into an Escherichia coli whole-cell biocatalyst. Through co-expression of the OmpF mutant with wild-type P450BM3 in the presence of decoy molecules, the yield of the whole-cell biotransformation of benzene could be considerably improved. Notably, with the decoy molecule C7AM-Pip-Phe the yield duodecupled from 5.7 % to 70 %, with 80 % phenol selectivity. The benzylic hydroxylation of alkyl- and cycloalkylbenzenes was also examined, and with the aid of decoy molecules, propylbenzene and tetralin were converted to 1-hydroxylated products with 78 % yield and 94 % (R) ee for propylbenzene and 92 % yield and 94 % (S) ee for tetralin. Our results suggest that both the decoy molecule and substrate traverse the artificial OmpF channel, synergistically boosting whole-cell bioconversions., (© 2021 Wiley-VCH GmbH.)

Nitric oxide (NO) reductase from the fungus Fusarium oxysporum is a P450-type enzyme (P450nor) that catalyzes the reduction of NO to nitrous oxide (N 2 O) in the global nitrogen cycle. In this enzymatic reaction, the heme-bound NO is activated by the direct hydride transfer from NADH to generate a short-lived intermediate ( I ), a key state to promote N-N bond formation and N-O bond cleavage. This study applied time-resolved (TR) techniques in conjunction with photolabile-caged NO to gain direct experimental results for the characterization of the coordination and electronic structures of I TR freeze-trap crystallography using an X-ray free electron laser (XFEL) reveals highly bent Fe-NO coordination in I , with an elongated Fe-NO bond length (Fe-NO = 1.91 Å, Fe-N-O = 138°) in the absence of NAD + TR-infrared (IR) spectroscopy detects the formation of I with an N-O stretching frequency of 1,290 cm -1 upon hydride transfer from NADH to the Fe 3+ -NO enzyme via the dissociation of NAD + from a transient state, with an N-O stretching of 1,330 cm -1 and a lifetime of ca. 16 ms. Quantum mechanics/molecular mechanics calculations, based on these crystallographic and IR spectroscopic results, demonstrate that the electronic structure of I is characterized by a singly protonated Fe 3+ -NHO •- radical. The current findings provide conclusive evidence for the N 2 O generation mechanism via a radical-radical coupling of the heme nitroxyl complex with the second NO molecule., Competing Interests: The authors declare no competing interest.

Cytochrome P450BM3 has long been regarded as a promising candidate for use as a biocatalyst, owing to its excellent efficiency for the hydroxylation of unactivated C-H bonds. However, because of its high substrate specificity, its possible applications have been severely limited. Consequently, various approaches have been proposed to overcome the enzyme's natural limitations, thereby expanding its substrate scope to encompass non-native substrates, evoking chemoselectivity, regioselectivity and stereoselectivity and enabling previously inaccessible chemical conversions. Herein, these approaches will be classified into three categories: (1) mutagenesis including directed evolution, (2) haem substitution with artificial cofactors and (3) use of substrate mimics, 'decoy molecules'. Herein, we highlight the representative work that has been conducted in above three categories for discussion of the future outlook of P450BM3 in green chemistry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

We report the enhanced cis- and enantioselective cyclopropanation of styrene catalysed by cytochrome P450BM3 in the presence of dummy substrates, i.e. decoy molecules. With the aid of the decoy molecule R-Ibu-Phe, diastereoselectivity for the cis diastereomers reached 91%, and the enantiomeric ratio for the (1S,2R) isomer reached 94%. Molecular dynamics simulations underpin the experimental data, revealing the mechanism of how enantioselectivity is controlled by the addition of decoy molecules.

We prepared enzyme-immobilized hydrogels and investigated the effects of the cross-linking density and polymer properties on their oxidation reaction rate. The oxidation rate of enzyme-immobilized hydrogels increased as the cross-linking density in the hydrogels increased. In addition, we controlled the oxidation rate using hydrogels exhibiting an appropriate interaction with a decoy molecule in the hydrogel.

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure-based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N-abietoyl-l-tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild-type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near-atomic resolution of 1.22 Å. Using these crystals as seeds in a cross-microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem-domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial cytochrome P450s (P450s) are at the focus of attention as potential biocatalysts for applications in green synthetic chemistry, as they possess high activity for the hydroxylation of inert substrate C-H bonds. The high activity of bacterial P450s, such as P450BM3, is chiefly due to their high substrate specificity, and consequently, the catalytic activity of P450BM3 toward non-native substrates is very low, limiting the utility of bacterial P450s as biocatalysts. To enable oxidation of non-native substrates by P450BM3 without any mutagenesis, we have developed a series of "decoy molecules", inert dummy substrates, with structures that resemble those of the native substrates. Decoy molecules fool P450BM3 into generating the active species, so-called Compound I, enabling the catalytic oxidation of non-native substrates other than fatty acids. Perfluorinated carboxylic acids (PFCs) serve as decoy molecules to initiate the activation of molecular oxygen in the same manner as long-alkyl-chain fatty acids, due to their structural similarity, and induce the generation of Compound I, but, unlike the native substrates, PFCs are not oxidizable by Compound I, allowing the hydroxylation of non-native substrates, such as gaseous alkanes and benzene. The catalytic activity for non-native substrate hydroxylation was significantly enhanced by employing second generation decoy molecules, PFCs modified with amino acids (PFC-amino acids). Cocrystals of P450BM3 with PFC9-Trp revealed clear electron density in the fatty-acid-binding channel that was readily assigned to PFC9-Trp. The alkyl chain terminus of PFC9-Trp does not reach the active site owing to multiple hydrogen bonding interactions between the carboxyl and carbonyl groups of PFC9-Trp and amino acids located at the entrance of the substrate binding channel of P450BM3 that fix it in place. The remaining space above the heme after binding of PFC9-Trp can be utilized to accommodate non-native substrates. Further developments revealed that third generation decoy molecules, N-acyl amino acids, such as pelargonoyl-l-phenylalanine (C9-Phe), can serve as decoy molecules, indicating that the rationale "fluorination is required for decoy molecule function" can be safely discarded. Diverse carboxylic acids including dipeptides could now be exploited as building blocks, and a library of decoy molecules possessing diverse structures was prepared. Among the third-generation decoy molecules examined N-enanthyl-l-proline modified with l-phenylalanine (C7-Pro-Phe) afforded the maximum turnover rate for benzene hydroxylation. The structural diversity of third-generation decoy molecules was also utilized to control the stereoselectivity of hydroxylation for the benzylic hydroxylation of Indane, showing that decoy molecules can alter stereoselectivity. As both the catalytic activity and enantioselectivity are dependent upon the structure of the decoy molecules, their design allows us to regulate reactions catalyzed by wild-type enzymes. Furthermore, decoy molecules can also activate intracellular P450BM3, allowing the use of E. coli expressing wild-type P450BM3 as an efficient whole-cell bioreactor. It should be noted that Mn-substituted full-length P450BM3 (Mn-P450BM3) is also active for the hydroxylation of propane in which the regioselectivity diverged from that of Fe-P450BM3. The results summarized in this Account represent good examples of how the reactive properties of P450BM3 can be controlled for the monooxygenation of non-native substrates in vitro as well as in vivo to expand the potential of P450BM3.

2,3',4,4',5-Pentachlorobiphenyl (CB118) is one of the most abundant polychlorinated biphenyl (PCB) congeners in the environment, and perfluoroalkyl acids, including perfluorocarboxylic acids (PFCAs), are widely distributed in the environment. Although CB118 and perfluoroalkyl acids are present in all humans and biota, effects in the metabolic fate of CB118 leading to toxicity change are unclear. P450BM3, which is isolated from the soil bacterium Bacillus megaterium, metabolized CB118 to three different hydroxylated pentachlorobiphenyls (M1-M3). M2 was identified as 4'-OH-2,3',4,5,5'-pentachlorobiphenyl. These reactions were promoted by the presence of PFCAs, and perfluorooctanoic acid (PFCA-C8) was the most effective for accelerating these reactions among PFCAs with different carbon chain length. The production rate of M2 was accelerated by 25-times using PFCA-C8. Furthermore, the docking models of P450BM3 with CB118 and PFCAs revealed that the conformational changes of the substrate-binding cavity of P450BM3 after binding of PFCAs to P450BM3 were important for selective production of CB118 metabolites. This study leads to the clarification of the different metabolic fates of PCBs under complex contamination with PFCAs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Haem substitution is an effective approach to tweak the function of haemoproteins. Herein, we report a facile haem substitution method for self-sufficient cytochrome P450BM3 (CYP102A1) from Bacillus megaterium utilising the transpeptidase Sortase A from Staphylococcus aureus. We successfully constructed Mn-substituted BM3 and investigated its catalytic activity.

We report a unique strategy for the development of a H 2 O 2 -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 H 2 O 2 . This system affords the best peroxygenase activity for the epoxidation of styrene, sulfoxidation of thioanisole, and hydroxylation of ethylbenzene among those P450-H 2 O 2 system previously reported. This work provides the first example of the activation of the normally H 2 O 2 -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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The wild-type cytochrome P450 (CYP) monooxygenase enzyme CYP102A1 (P450Bm3) has low activity for cycloalkane oxidation. The oxidation of these substrates by variants of this enzyme in combination with perfluorinated decoy molecules (PFCs) was investigated to improve productivity. The use of rate accelerating variants, which have mutations located outside of the substrate binding pocket as well as an active site variant of CYP102A1 (A74G/F87V/L188Q) all enhanced cycloalkane oxidation (C5 to C10). The addition of the decoy molecules to the wild-type and the rate accelerating mutants of CYP102A1 boosted the substrate oxidation rates even further. However, the levels of cycloalkanol product decreased with the larger alkanes when the decoy molecules were used with the variant A74G/F87V/L188Q, which contained mutations within the substrate binding pocket. For the majority of the enzymes and PFC decoy molecule combinations the highest levels of oxidation were obtained with cyclooctane. When larger second generation decoy molecules, based on modified amino acids were utilised there was a significant improvement in the oxidation of the smaller cycloalkanes by the wild-type enzyme and one other variant. This resulted in significant improvements in biocatalytic oxidation of cyclopentane and cyclohexane. However, the use of these optimised decoy molecules did not significantly improve cycloalkane oxidation over the fluorinated fatty acid derivatives when combined with the best rate accelerating variant, R47L/Y51F/I401P. Overall our approach enabled the cycloalkanes to be oxidised 300- to 8000-fold more efficiently than the wild-type enzyme at product formation rates in excess of 500 and up to 1700 nmol·nmol-CYP -1 ·min -1 ., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min -1  P450BM3 -1 and 40 200 P450BM3 -1 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cytochrome P450SPα (P450SPα) and cytochrome P450BSβ (P450BSβ) belonging to the CYP152 family of enzymes (CYP152s) can utilize H2O2 efficiently as an oxidant for the generation of compound I. Although P450SPα and P450BSβ have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as "decoy molecules." Although we had assumed that acetic acid did not serve as a decoy molecule, P450SPα and P450BSβ efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450BSβ in the presence of 1 M acetate anion reached 590 ± 30 min(-1).

Cytochrome P450s (P450s) catalyze the NAD(P)H/O2-dependent monooxygenation of less reactive organic molecules under mild conditions. The catalytic activity of bacterial P450s is very high compared with P450s isolated from animals and plants, and the substrate specificity of bacterial P450s is also very high. Accordingly, their catalytic activities toward nonnative substrates are generally low especially toward small hydrocarbons. However, mutagenesis approaches have been very successful for engineering bacterial P450s for the hydroxylation of small hydrocarbons. On the other hand, "decoy" molecules, whose structures are very similar to natural substrates, can be used to trick the substrate recognition of bacterial P450s, allowing the P450s to catalyze oxidation reactions of nonnative substrates without any substitution of amino acid residues in the presence of decoy molecules. Thus, the hydroxylation of small hydrocarbons such as ethane, propane, butane and benzene can be catalyzed by P450BM3, a long-alkyl-chain hydroxylase, using substrate misrecognition of P450s induced by decoy molecules. Furthermore, a number of H2O2-dependent bacterial P450s can catalyze the peroxygenation of a variety of nonnative substrates through a simple substrate-misrecognition trick, in which catalytic activities and enantioselectivity are dependent on the structure of decoy molecules.

Cytochromes P450 (P450s) catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. By contrast with the general reductive oxygen activation pathway of P450s, an H2O2-shunt pathway does not require any supply of electrons and protons for the generation of a highly reactive intermediate (compound I). Because the low cost of H2O2 allows us to use it in industrial-scale synthesis, the H2O2-shunt pathway is an attractive process for monooxygenation reactions. This review focuses on the P450-catalyzed monooxygenation of organic molecules using H2O2 as the oxidant.

Cytochrome P450(BSβ), a H(2)O(2)-dependent cytochrome P450 catalyzing the hydroxylation of long-alkyl-chain fatty acids, lacks the general acid-base residue around the heme, which is indispensable for the efficient generation of the active species using H(2)O(2). On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450(BSβ), it was suggested that the role of the general acid-base function was provided by the carboxylate group of fatty acids. The participation of the carboxylate group of the substrate was supported by the fact that cytochrome P450(BSβ) can catalyze oxidations of nonnatural substrates such as styrene and ethylbenzene in the presence of a series of short-alkyl-chain carboxylic acids as a dummy molecule of fatty acid. We refer to a series of short-alkyl-chain carboxylic acids as a "decoy molecule". As shown here, we have clarified the crystal structure of the decoy-molecule-bound form and elucidated that the location of its carboxylate group is virtually the same as that of palmitic acid in the heme cavity, indicating that the carboxylate group of the decoy molecule serves as the general acid-base catalyst. This result further confirms that the role of the acid-base function is satisfied by the carboxylate group of the substrates. In addition, the structure analysis of the substrate-free form has clarified that no remarkable structural change is induced by the binding of the decoy molecule as well as fatty acid. Consequently, whether the carboxylate group is positioned in the active site provides the switching mechanism of the catalytic cycle of cytochrome P450(BSβ).

Aromatic C-H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450BSbeta (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig's blue. Russig's blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements.