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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. [ABSTRACT FROM AUTHOR]

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 kcat 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 (kcat/Km = 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Keywords: Asymmetric Catalysis; Crystallography; Cytochrome P450; Decoy Molecules; Reaction Mechanisms EN Asymmetric Catalysis Crystallography Cytochrome P450 Decoy Molecules Reaction Mechanisms 1 1 1 03/15/23 20230320 NES 230320 B Enzymes b . In their Research Article (e202215706), Hiroshi Sugimoto, Osami Shoji et al. report the crystal structure of cytochrome P450BM3 mimicking the state of the enzyme in the moment preceding epoxidation. Frontispiece: A Compound I Mimic Reveals the Transient Active Species of a Cytochrome P450 Enzyme: Insight into the Stereoselectivity of P450-Catalysed Oxidations. [Extracted from the article]

In their Research Article (e202215706), Hiroshi Sugimoto, Osami Shoji et al. report the crystal structure of cytochrome P450BM3 mimicking the state of the enzyme in the moment preceding epoxidation. Keywords: Asymmetrische Katalyse; Kristallografie; Reaktionsmechanismen; Täuschmoleküle; Zytochrom P450 DE Asymmetrische Katalyse Kristallografie Reaktionsmechanismen Täuschmoleküle Zytochrom P450 1 1 1 03/15/23 20230320 NES 230320 B Enzymes b . [Extracted from the article]

Playing tricks on enzymes: Direct hydroxylation of benzene to phenol was catalyzed by wild‐type P450BM3 in the presence of perfluorinated carboxylic acids as decoy molecules. The catalytic turnover rate reached 120 min−1 per P450. The selectivity towards phenol production was very high and no overoxidation products were detected. [ABSTRACT FROM AUTHOR]

Keywords: cytochrome P450; whole-cell biocatalyst; decoy molecules; benzene hydroxylation; stereoselective oxidation EN cytochrome P450 whole-cell biocatalyst decoy molecules benzene hydroxylation stereoselective oxidation 1 1 1 02/03/22 20220207 NES 220207 B A loop deletion mutant b of outer membrane protein F (OmpF) that facilitates the cellular uptake of decoy molecules into I Escherichia coli i has been developed. Inside Cover: Designer Outer Membrane Protein Facilitates Uptake of Decoy Molecules into a Cytochrome P450BM3-Based Whole-Cell Biocatalyst (Angew. Cytochrome P450, whole-cell biocatalyst, decoy molecules, benzene hydroxylation, stereoselective oxidation. [Extracted from the article]

A correction to the article "Aromatic C–H bond hydroxylation by P450 peroxygenases: a facile colorimetric assay for monooxygenation activities of enzymes based on Russig’s blue formation" that was published in the previous issue of the journal is presented.

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. [ABSTRACT FROM AUTHOR]

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. Decoy molecules were shown to be compatible for use with both CYP102A5 and CYP102A7. Both CYP102A5 and CYP102A7 displayed a unique and unexpected decoy molecule preference. The crystal structure of CYP102A5 was solved at a resolution of 2.8 Å. [Display omitted] • Decoy molecules were shown to be compatible for use with both CYP102A5 and A7. • Both CYP102A5 and A7 displayed a unique and unexpected decoy molecule preference. • Decoy molecules were found to bind in a two-step fashion to CYP102A5 and A7 that was not observed for A1. • We report the first structure of CYP102A5 at a resolution of 2.8 Å. • The structure of CYP102A5 reveals a potential binding site that differs significantly from the CYP102A1 reference system. [ABSTRACT FROM AUTHOR]