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Abstract Perfluorinated decoy molecules based on a combination of fatty and amino acids were used to enhance hydroxylation, epoxidation and sulfoxidation reactions of P450Bm3. The combination of amino acid derived second generation decoy molecules, with the rate accelerating variant R19 (R47L/Y51F/H171L/Q307H/N319Y) displayed the highest oxidation rates. Mutation of Thr268 to Glu (Bm3TE) converted the heme domain to a H 2 O 2 utilising peroxygenase. This Bm3TE variant displayed significant peroxygenase activity towards all the substrates tested with a preference for methylthiobenzene sulfoxidation. However, the addition of decoy molecules did not improve the efficiency of this variant. Graphical abstract Unlabelled Image Highlights • Decoy molecules were used to increase the productivity of P450 holoenzyme variants. • Hydroxylation and epoxidation reactions were improved dramatically as was sulfoxidation. • A heme domain peroxygenase variant (Thr268Glu) catalysed P450 activity with H 2 O 2. • The peroxygensae catalysed sulfoxidation > epoxidation > benzylic C H hydroxylation. • Decoy molecules did not improve peroxygenase productivity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The heme of hydrogen peroxide-dependent cytochrome P450BSβ (P450BSβ) was removed by apomyoglobin under mild conditions to give apo-P450BSβ without the need for acidic conditions and organic solvents. The circular dichroism spectrum of the apo-P450BSβ was essentially identical to that of holo-P450BSβ, showing a small structural change resulting from the removal of heme using apomyoglobin. The apo-P450BSβ was reconstituted with hemin or manganese protoporphyrin IX (MnPPIX), and the resulting reconstituted P450BSβ catalyzed the one-electron oxidation of guaiacol using hydrogen peroxide as an oxidant. A higher catalytic activity was observed for P450BSβ reconstituted with MnPPIX when meta-chloroperoxybenzoic acid (mCPBA) was used as the oxidant. [ABSTRACT FROM AUTHOR]

[Display omitted] • The critical function of haem-acquisition system of gram-negative bacterial pathogens. • Artificial haem-acquisition protein HasAs harbouring synthetic metal complexes and their crystal structure analysis. • Antimicrobial applications of HasA capturing synthetic complexes. • Chemical applications of artificial-HasAs including catalytic utilities. Certain bacterial pathogens secrete haemophores, i.e. extracellular haem-uptake proteins, which acquire haem as a vital source of iron in the iron-devoid environment of their hosts. The haem-binding site of haemophores is highly exposed to the protein surface, enabling them to pirate haem from other haem-containing proteins (haemoproteins). This review will discuss the hidden ability of a representative haemophore, namely the haem-acquisition system protein A (HasA) secreted by Pseudomonas aeruginosa , which can accommodate a diverse assortment of synthetic metal complexes possessing extreme insolubility and/or high steric bulk in its haem-binding site. The replacement of haem with similar synthetic analogues is an already well established and powerful means to change the function of select haemoproteins; however, usable complexes are severely limited by the high selectivity of haemoproteins for haem. This review will introduce how combining the haem-replacement approach with HasA can open the door for the biological application of diverse synthetic metal complexes that were previously incompatible with biological systems. [ABSTRACT FROM AUTHOR]

Enzyme performance can be improved using decoy molecules or engineered variants to accelerate the activity without affecting selectivity. Here we combine a rate accelerator variant of cytochrome P450Bm3 with decoy molecules to enhance the oxidation activity of a range of small organic molecules. This combined approach offers superior biocatalytic efficiency without modifying the product distribution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

To bring out the potential of wild-type cytochrome P450s, we have developed a series of 'decoy molecules' to change their high substrate specificity without any mutagenesis. Decoy molecules are inert dummy substrates with structures that are very similar to those of natural substrates. The decoy molecules force long-alkyl-chain fatty acid hydroxylases (P450BSβ, P450SPα, and P450BM3) to generate the active species and to catalyze oxidation of various substrates other than fatty acids. Interestingly, the catalytic activity was highly dependent on the structure of decoy molecules. Furthermore, the enantioselectivity of reactions catalyzed by P450BSβ and P450SPα was also dependent on the structure of decoy molecules. The decoy molecule system allows us to control reactions catalyzed by wild-type enzymes by designing decoy molecules. [ABSTRACT FROM AUTHOR]

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 HO-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 HO allows us to use it in industrial-scale synthesis, the HO-shunt pathway is an attractive process for monooxygenation reactions. This review focuses on the P450-catalyzed monooxygenation of organic molecules using HO as the oxidant. [ABSTRACT FROM AUTHOR]

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasAp) can capture several synthetic metal complexes other than heme. The crystal structures of HasAp harboring synthetic metal complexes revealed only small perturbation of the overall HasAp structure. An inhibitory effect upon heme acquisition by HasAp bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasAp bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasAp as an iron source. [ABSTRACT FROM AUTHOR]

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasAp) can capture several synthetic metal complexes other than heme. The crystal structures of HasAp harboring synthetic metal complexes revealed only small perturbation of the overall HasAp structure. An inhibitory effect upon heme acquisition by HasAp bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasAp bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasAp as an iron source. [ABSTRACT FROM AUTHOR]

Tricks mit Enzymen: Die direkte Hydroxylierung von Benzol zu Phenol wurde durch Wildtyp ‐ P450BM3 in der Gegenwart von perfluorierten Carbonsäuren als Ködermolekülen katalysiert. Der katalytische Umsatz erreichte 120 min−1 pro P450. Die Selektivität für die Phenolbildung war sehr hoch, und Produkte einer Überoxidation wurden nicht nachgewiesen. [ABSTRACT FROM AUTHOR]

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]

The stereoselective epoxidation of styrene was catalyzed by H2O2-dependent cytochrome P450SPα in the presence of carboxylic acids as decoy molecules. The stereoselectivity of styrene oxide could be altered by the nature of the decoy molecules. In particular, the chirality at the α-positions of the decoy molecules induced a clear difference in the chirality of the product: ( R)-ibuprofen enhanced the formation of ( S)-styrene oxide, whereas ( S)-ibuprofen preferentially afforded ( R)-styrene oxide. The crystal structure of an ( R)-ibuprofen-bound cytochrome P450SPα (resolution 1.9 Å) revealed that the carboxylate group of ( R)-ibuprofen served as an acid-base catalyst to initiate the epoxidation. A docking simulation of the binding of styrene in the active site of the ( R)-ibuprofen-bound form suggested that the orientation of the vinyl group of styrene in the active site agreed with the formation of ( S)-styrene oxide. [ABSTRACT FROM AUTHOR]

Cytochrome P450SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H2O2-dependent P450. P450SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the Ca of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450SPα. Mutations at the active site and the F-G loop of P450SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450BSβ. [ABSTRACT FROM AUTHOR]

Cytochrome P450, a HO-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 HO. On the basis of the crystal structure of the palmitic acid bound form of cytochrome P450, 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 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. [ABSTRACT FROM AUTHOR]

omatic C–H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450 (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. [ABSTRACT FROM AUTHOR]

Abstract: Novel imidazolylethynyl-zinc-porphyrin 1a and its meso,meso-linked bisporphyrin 5M were synthesized effectively by the reaction of the corresponding bromoporphyrins and 2-imidazolylethyne in the presence of palladium-arsenic catalyst. The complementary coordination of monomer 1a into dimer 2a was observed by 1H NMR and UV–Vis spectroscopy. Self-association constant of 1a to 2a in CHCl3 (including 0.5% ethanol) was determined as 1.84×107 M−1 by UV–Vis titration of 2a with N-methylimidazole. UV–Vis absorption and fluorescence spectra of 1a, 2a, monomer 5M, and its polymer 5P were compared. [Copyright &y& Elsevier]

Ferrocene-bridged trisporphyrin (2) was synthesized by two-steps condensation of corresponding aldehydes and dipyrromethanes, and its self-assembling behavior based on the complementary coordination motif of imidazolylporphyrinatozinc(II) was investigated in conjunction with hinge-like flexibility given by freely rotating cyclopentadienyl rings of ferrocene connector. Ferrocene-bridged trispoiphyrin (2) spontaneously and exclusively generated the dimeric ring (7) upon simple zinc(II) insertion, indicating that the freely rotating hinge connector favored the smallest ring formation. Taking advantage of the unique hinge-like flexibility of ferrocene, we attempted to transform the dimer ring into a mixture of porphyrin macrocycles by reorganizing the structure cleaved once by pyridine. A series of porphyrin macrocycles from trimer to decamer can be separated into its components by preparative gel permeation chromatograms. Macrocycles obtained are kept stable in the absence of coordinating solvents. On the other hand, they were easily transformed to the dimer ring in the presence of coordinating solvents such as methanol, showing that the transformation is completely reversible and can be controlled by the choice of the solvent system. A series of porphyrin macrocycles was confirmed via covalent linking of each complementary coordination dimer pair by metathesis reaction in the presence of Grubbs's catalyst. The coordination behavior of the bidentate ligands with different spacer lengths toward the dimer ring revealed that only the bidentate ligand (15) with a spacer length that matched the facing central porphyrins was selectively accommodated inside the ring. Coordination assembled flexible rings with tunable cavities and multiple coordination sites will be used as versatile hosts for a wide variety of guest molecules. [ABSTRACT FROM AUTHOR]

Poly[ N 5 -( R and S )-1-(1-pyrenyl)ethyl- l -glutamines] ( 1 and 2 ) were prepared by condensation of poly( l -glutamic acid) with optically resolved amines. In solution, these polymers, 2 in particular, gave large circular dichroism (CD) indicative of exciton coupling among the side-chain pyrene chromophores. When compared with the corresponding polymer with achiral side groups, i.e. poly(1-pyrenylmethyl- l -glutamine) ( 3 ), 1 and 2 not only gave much stronger CD, but also gave much reduced excimer emission with a significant hypsochromic shift of emission maximum. The highly controlled orientation of the side-chain chromophores is apparently brought about by the specific steric interactions among the bulky chiral side chains along the helical main chain. [ABSTRACT FROM AUTHOR]

We report the directed evolution of cytochrome P450BM3 to efficiently utilize the bacterial quorum sensing signalling molecule N‐decanoyl homoserine lactone (C10‐HSL) as an effective decoy molecule. This represents the first important step in our endeavor to develop of a self‐sufficient decoy‐molecule system in whole‐cells that only necessitates the addition of culture medium and substrate to realize the hydroxylation of non‐native substrates. Following five rounds of directed evolution, mutant P450BM3, in the presence of C10‐HSL, catalyzed the hydroxylation of benzene at a rate of 475 min−1, the highest turnover rate recorded for any P450 enzyme, and achieving a 46% yield in a whole‐cell reaction system. High‐resolution X‐ray crystal structure analysis of a series of mutants narrates the directed evolution process, revealing how C10‐HSL is fixed in the binding pocket to permit binding of non‐native substrates. Finally, introduction of the C10‐HSL synthase gene ExpI into Escherichia coli, enabled the in situ production of C10‐HSL, realizing, for the first time, the hydroxylation of non‐native substrates without the need for the laborious synthesis and addition of decoy molecules. [ABSTRACT FROM AUTHOR]

Abstract: The enantioselectivity in the sulfoxidation of thioanisole catalyzed by cytochrome P450BSβ with a decoy molecule, a dummy molecule of the natural substrate, can be inverted by changing the structure of the decoy molecule. The methodology demonstrated herein shows the potential for controlling the stereoselectivity of biocatalysts without any mutagenesis. [Copyright &y& Elsevier]

Oligonucleotides represent powerful DNA-recognition tools, but the formation of undesirable "self-duplexes" becomes more probable with increasing DNA affinity. Herein, we have developed a modified nucleobase with "self-avoiding" properties. Facile methylation of guanine yields a cationic N7-methylguanine, which suppresses the formation of self-duplexes whilst improving DNA affinity through electrostatic interaction. [ABSTRACT FROM AUTHOR]

Hydroxylierung In der Zuschrift auf S. 10460 beschreiben O. Shoji, Y. Watanabe et al. die Verwendung von bestimmten Aminosäurederivaten für die Aktivierung von Cytochrom P450BM3 zur direkten Hydroxylierung von Benzol zu Phenol. [ABSTRACT FROM AUTHOR]

In this article the authors have observed single molecular STM images of the coordination-assembled porphyrin macrocycles deposited on a metal surface by the pulse injection method and succeeded in visualizing the extremely large porphyrin macroring consisting of 30 porphyrins with hollow structure. Covalent linking of the coordination pairs was effective to reinforce the supramolecular structure on a metal surface. This strategy will allow them to know the real structure of supramolecular assemblies by scanning tunneling microscopy, rather than by NMR or X-ray crystal analysis. This will be applicable to a variety of supramolecular assemblies.

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 . [ABSTRACT FROM AUTHOR]

Das Hämaufnahmeprotein von P. aeruginosa (HasAp), mit der Form eines Fisches, nimmt auch verschiedene synthetische Eisenkomplexe, wie Eisen ‐ Salophene und Eisen ‐ Phthalocyanine, auf. In ihrer Zuschrift auf S. 2906 ff. zeigen O. Shoji, Y. Watanabe und Mitarbeiter, welche Auswirkungen die Bindung von HasAp an diese nichtnatürlichen Eisenkomplexe auf das Wachstum von P. aeruginosa und die HasAp ‐ vermittelte Eisenaufnahme hat. [ABSTRACT FROM AUTHOR]

The secreted heme acquisition protein of P. aeruginosa (HasAp), which is shaped like a fish, was found to be able to capture various synthetic iron complexes, such as iron–salophen and iron–phthalocyanine. In their Communication on page 2862 ff., O. Shoji, Y. Watanabe, and co‐workers demonstrate the effect of HasAp bound to these unnatural iron complexes on the growth of P. aeruginosa and show that HasAp bound to iron–phthalocyanine inhibits HasAp‐mediated iron acquisition. [ABSTRACT FROM AUTHOR]

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]

Das Erfassen der Struktur von Zytochrom‐P450‐Enzymen in flagranti ist für die Entwicklung von P450‐Biokatalysatoren von entscheidender Bedeutung, da die meisten der bisher erfassten Strukturen in einer präkatalytischen Konformation gefangen sind. Das Herzstück der P450‐Katalyse ist Verbindung I (Vbd I), ein kurzlebiges, hochreaktives Zwischenprodukt, dessen widerspenstige Art die Mehrheit der Versuche vereitelt hat, katalytisch bedeutsame Konformationen von P450 zu erfassen. Wir zeigen die Kristallstruktur von P450BM3, die den Zustand genau im Momente vor der Epoxidierung nachahmt, der mit den experimentell ermittelten Stereoselektivitäten perfekt übereinstimmt. Die Kristallisation wurde durch Bindung des stabilen Vbd‐I‐Analogons Oxomolybdänmesoporphyrin IX an P450BM3 in Gegenwart Styrols erreicht. Die relative Ausrichtung Styrols zur Molybdän‐Sauerstoff‐Spezies in den Kristallstrukturen wirft ein Licht auf die Dynamik, die an der Drehung Styrols beteiligt ist, um seine Vinylgruppe Vbd I darzubieten. Diese Methode dient als leistungsfähiges Instrument zur Vorhersage und Modellierung der Stereoselektivität von P450‐Reaktionen. [ABSTRACT FROM AUTHOR]

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.

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]

Using artificial hemes for the reconstruction of natural heme proteins represents a fascinating approach to enhance the bioactivity of the latter. We report the synthesis of various metal 5‐oxaporphyrinium cations as cofactors, and a cobalt 5‐oxaporphyrinium cation was successfully incorporated into the heme‐acquisition protein (HasA) secreted by Pseudomonas aeruginosa. We hypothesize that the oxaporphyrinium cation strongly binds to the HasA‐specific outer membrane receptor (HasR) due to its cationic charge, which prevents the subsequent acquisition of heme. In fact, the reconstructed HasA inhibited the growth of Pseudomonas aeruginosa and even of multidrug‐resistant P. aeruginosa. [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]

Using artificial hemes for the reconstruction of natural heme proteins represents a fascinating approach to enhance the bioactivity of the latter. We report the synthesis of various metal 5‐oxaporphyrinium cations as cofactors, and a cobalt 5‐oxaporphyrinium cation was successfully incorporated into the heme‐acquisition protein (HasA) secreted by Pseudomonas aeruginosa. We hypothesize that the oxaporphyrinium cation strongly binds to the HasA‐specific outer membrane receptor (HasR) due to its cationic charge, which prevents the subsequent acquisition of heme. In fact, the reconstructed HasA inhibited the growth of Pseudomonas aeruginosa and even of multidrug‐resistant P. aeruginosa. [ABSTRACT FROM AUTHOR]

Wir berichten über eine OmpF‐Schleifenlöschungsmutante, welche die Zellaufnahme externer Additiva in einen Escherichia‐coli‐Ganzzellbiokatalysator verbessert. Durch die Koexprimierung der OmpF‐Mutante mit Wildtyp‐P450BM3 in Gegenwart von Täuschmolekülen konnte die Ausbeute der Ganzzellbiotransformation Benzols erheblich gesteigert werden. Insbesondere mit dem Täuschmolekül C7AM‐Pip‐Phe verzwölffachte sich die Ausbeute von 5.7 % auf 70 % mit einer Phenolselektivität von 80 %. Die Untersuchung der benzylischen Hydroxylierung von Alkyl‐ und Zykloalkylbenzolen ergab, dass mit Hilfe von Täuschmolekülen Propylbenzol und Tetralin jeweils mit 78 % Ausbeute und einer Selektivität von 94 % (R) ee für Propylbenzol and 92 % Ausbeute und einer Selektivität von 94 % (S) ee für Tetralin in ihre 1‐Hydroxylierungsprodukte überführt wurden. Unsere Ergebnisse deuten darauf hin, dass sowohl das Täuschmolekül als auch das Substrat den künstlichen OmpF‐Kanal durchqueren und dadurch die Ganzzellbiotransformation in ganzen Zellen synergetisch verstärken. [ABSTRACT FROM AUTHOR]

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]

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. [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]

Die Kristallisation der Hämdomäne von CYP102A1 (P450BM3) kann nach Modifizierung eine Herausforderung sein. Dennoch sind solche Kristallstrukturen unentbehrlich für die wirksame strukturbezogene Entwicklung von P450BM3 als Biokatalysator. Es wurde gezeigt, dass das Abietanditerpenderivat N‐Abietoyl‐l‐tryptophan (AbiATrp) ein hervorragender Kristallisationsbeschleuniger ist. Die Nutzung von Kristallen der P450BM3‐Hämdomäne mit AbiATrp als Impfkristalle ermöglichte die rasche Mikrokristallisation bei der naszierende Kristalle innerhalb von Sekunden nach Impfung erschienen. Hierdurch wurde eine Auswahl an Kristallen der P450BM3‐Hämdomäne erhalten, die bisher nicht kristallisierbare Täuschmoleküle, diverse künstliche Metalloporphyrine, welche verschiedenartige Liganden binden, sowie zusätzlich stark getaggte Hämdomänevarianten enthalten (37 zusätzliche Aminosäuren). Manche der Strukturen könnten als Modellverbindungen verschiedener Stadien des katalytischen Zyklus von P450BM3 genutzt werden. [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]

A new type of high‐pressure reactor based on liquid‐flow pressurisation using a HPLC pump has been developed. This high‐pressure reactor allows the easy and safe performance of reactions with gaseous alkanes under high‐pressures up to 10 MPa (100 atm), without the need for high‐pressure gas cylinders. The amount of substrate gas required for a single reaction is very small compared with reactions using a conventional autoclave, which, when using expensive substrate gasses, such as 13C‐labelled ethane, becomes critical. Employing this high‐pressure reactor in conjunction with cytochrome P450BM3 and the assistance of decoy molecules, the direct hydroxylation of gaseous alkanes was drastically improved. At 5 MPa the TOF of propane hydroxylation increased 10‐fold, reaching 2200 min−1. Hydroxylation of ethane was also substantially accelerated at 5 MPa, reaching a TOF of 28 min−1. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • Bacterial P450BM3 metabolized CB118 to hydroxylated metabolites. • Hydroxylation of CB118 was promoted by the existence of PFCAs. • The size of the substrate-binding cavity of P450BM3 was changed by binding PFCAs. • Metabolic fates of PCBs under complex contamination with PFCAs may be different. [ABSTRACT FROM AUTHOR]

Abstract: Ein Escherichia‐coli‐Ganzzellbiokatalysator für die direkte Hydroxylierung von Benzol zu Phenol wurde entwickelt. Durch Zugabe von Aminosäurederivaten als Täuschmoleküle zum Kulturmedium konnte in E. coli exprimiertes Wildtyp‐Zytochrom P450 (P450BM3) aktiviert werden und nichtnative Substrate, ohne den Bedarf eines NADPH‐Zusatzes, hydroxylieren. In Gegenwart von N‐Heptyl‐ l‐prolyl‐ l‐phenylalanin (C7‐Pro‐Phe) als Täuschmolekül erreichte die Phenolausbeute 59 %. Es wurde gezeigt, dass Täuschmoleküle, vorzugsweise nichtfluorierte, das Zytosol von E. coli erreichen, wodurch intrazellulärem P450BM3 katalytische Aktivität für die In‐vivo‐Oxyfunktionalisierung nichtnativer Substrate verliehen wird. [ABSTRACT FROM AUTHOR]

Abstract: An Escherichia coli whole‐cell biocatalyst for the direct hydroxylation of benzene to phenol has been developed. By adding amino acid derivatives as decoy molecules to the culture medium, wild‐type cytochrome P450BM3 (P450BM3) expressed in E.coli can be activated and non‐native substrates hydroxylated, without supplementing with NADPH. The yield of phenol reached 59 % when N‐heptyl‐ l‐prolyl‐ l‐phenylalanine (C7‐Pro‐Phe) was employed as the decoy molecule. It was shown that decoy molecules, especially those lacking fluorination, reached the cytosol of E. coli, thus imparting in vivo catalytic activity for the oxyfunctionalisation of non‐native substrates to intracellular P450BM3. [ABSTRACT FROM AUTHOR]

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. [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]