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Cytochrome P450s are an important group of enzymes catalyzing hydroxylation, and epoxidations reactions. In this work we describe the characterization of the CinA–CinC fusion enzyme system of a previously reported P450 using genetically fused heme (CinA) and FMN (CinC) enzyme domains from Citrobacter braaki. We observed that mixing individually inactivated heme (-) with FMN (-) domain in the CinA-10aa linker - CinC fusion constructs results in recovered activity and the formation of (2S)-2β-hydroxy,1,8-cineole (174 µM), a similar amount when compared to the fully functional fusion protein (176 µM). We also studied the effect of the fusion linker length in the activity complementation assay. Our results suggests an intermolecular interaction between heme and FMN parts from different CinA–CinC fusion protein similar to proposed mechanisms for P450 BM3 on the other hand, linker length plays a crucial influence on the activity of the fusion constructs. However, complementation assays show that inactive constructs with shorter linker lengths have functional subunits, and that the lack of activity might be due to incorrect interaction between fused enzymes. [ABSTRACT FROM AUTHOR]

Just like the drugs themselves, their metabolites have to be evaluated to succeed in a drug development and approval process. It is therefore essential to be able to predict drug metabolism and to synthesise sufficient metabolite quantities for further pharmacological testing. This study evaluates the possibility of using in vitro biotransformations to solve both these challenges in the case of testosterone as a representative component for steroids. The application of cells of Pichia pastoris with expressed membrane‐associated human liver cytochrome P450 enzyme (P450) 3A4 in two cycles of a preparative‐scale bioreactor experiment enabled the isolation of the common metabolites 6β‐hydroxytestosterone and 6β‐hydroxyandrostenedione on a 100 mg scale. Side‐product formation caused by enzymes intrinsic to P. pastoris was reduced. In addition more polar testosterone metabolites formed by a P450 3A4‐catalysed bioconversion, than the known mono‐hydroxylated ones, are reported and 6‐dehydro‐15β‐hydroxytestosterone as well as the di‐hydroxylated steroids 6β,16β‐dihydroxytestosterone, 6β,17β‐dihydroxy‐4‐androstene‐3,16‐dione and 6β,12β‐dihydroxyandrostenedione were isolated and verified by NMR analysis. Their respective biological significance remains to be investigated. Whole‐cell P450 catalysts expressed in P. pastoris qualify as a tool for the preparative‐scale synthesis of human metabolites. Biotransformation processes in combination with standard chemical procedures allow the isolation and characterisation even of minor drug metabolite products. [ABSTRACT FROM AUTHOR]

The direct hydroxylation of benzene to hydroquinone (HQ) under mild reaction conditions is a challenging task for chemical catalysts. Cytochrome P450 (CYP) monooxygenases are known to catalyze the oxidation of a variety of aromatic compounds with atmospheric dioxygen. Protein engineering campaigns led to the identification of novel P450 variants, which yielded improvements in respect to activity, specificity, and stability. An effective screening strategy is crucial for the identification of improved enzymes with desired characteristics in large mutant libraries. Here, we report a first screening system designed for screening of P450 variants capable to produce hydroquinones. The hydroquinone quantification assay is based on the interaction of 4-nitrophenylacetonitrile (NpCN) with hydroquinones under alkaline conditions. In the 96-well plate format, a low detection limit (5 μM) and a broad linear detection range (5 to 250 μM) were obtained. The NpCN assay can be used for the quantification of dihydroxylated aromatic compounds such as hydroquinones, catechols, and benzoquinones. We chose the hydroxylation of pseudocumene by P450 BM3 as a target reaction and screened for improved trimethylhydroquinone (TMHQ) formation. The new P450 BM3 variant AW2 (R47Q, Y51F, I401M, A330P) was identified by screening a saturation mutagenesis library of amino acid position A330 with the NpCN assay. In summary, a 70-fold improved TMHQ formation was achieved with P450 BM3 AW2 when compared to the wild type (WT) and a 1.8-fold improved TMHQ formation compared to the recently reported P450 BM3 M3 (R47S, Y51W, A330F, I401M). [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) and their N- and O-containing derivatives (N-/O-PAHs) are environmental pollutants and synthetically attractive building blocks in pharmaceuticals. Functionalization of PAHs can be achieved via C-H activation by cytochrome P450 enzymes (e.g., P450 CYP3A4) in an environmentally friendly manner. Despite its broad substrate scope, the contribution of CYP3A4 to metabolize common PAHs in humans was found to be small. We recently showcased the potential of CYP3A4 in whole-cell biocatalysis with recombinant yeast Komagataella phaffii (Pichia pastoris) catalysts for the preparative-scale synthesis of naturally occurring metabolites in humans. In this study, we aimed at exploring the substrate scope of CYP3A4 towards (N-/O)-PAHs and conducted a bioconversion experiment at 10 L scale to validate the synthetic potential of CYP3A4 for the preparative-scale production of functionalized PAH metabolites. Hydroxylated products were purified and characterized using HPLC and NMR analysis. In total, 237 mg of fluorenol and 48 mg of fluorenone were produced from 498 mg of fluorene, with peak productivities of 27.7 μmol/L/h for fluorenol and 5.9 μmol/L/h for fluorenone; the latter confirmed that CYP3A4 is an excellent whole-cell biocatalyst for producing authentic human metabolites. [ABSTRACT FROM AUTHOR]

Cytochrome P450 BM3 monooxygenase from Bacillus megaterium and its variants are promising catalysts for organic synthesis. Aiming at the identification of variants for selective hydroxylation of functionalised aromatic compounds, the double mutant F87A L188C showed remarkably improved catalytic activity towards a set of tested toluene derivatives. The apparent catalytic efficiency of this variant towards the model substrate methyl 2-methoxy-3-methylbenzoate was 63.6 s−1 M−1, which is 535-fold higher compared to that of wild-type BM3. Furthermore, the double mutant selectively catalysed the benzylic hydroxylation of numerous toluene derivatives, especially in the presence of carbonyl- or carboxyl-functions that are directly attached to the aromatic ring. Preparative scale conversion resulted in efficient production of methyl 3-(hydroxymethyl)-2-methoxybenzoate (73% yield) which proved that F87A L188C is a suitable, efficient and sustainable catalyst for the introduction of benzylic hydroxyl groups in general. [ABSTRACT FROM AUTHOR]

Cytochrome P450s are synthetically attractive hydroxylation catalysts. For cell-free applications, a constant supply of NAD(P)H can be very costly. Mediators such as Zn/Co(III)sep can be an alternative cofactor system to NAD(P)H. Several mutants of cytochrome P450 BM3 with improved electron transfer rate to Zn/Co(III)sep have been obtained in our group. P450 BM3 M7 (F87A V281G M354S R471C A1011T S1016G Q1022R) was immobilized on DEAE-650S, further entrapped with k-carrageenan together with zinc dust which function as electron source and catalase which removes produced hydrogen peroxide instantly. Immobilized P450 BM3 M7 were treated with 0.05% ( v/ v) glutaraldehyde to enhance operational stability. P450 BM3 M7 retained around 76% of its activity and conversions stayed above 80% in 10 batch cycles, indicating a high stability of immobilized P450 BM3 M7. To explore the synthetic potential, a small-scale bioreactor was developed to investigate the stability and efficiencies of P450 BM3 M9 (R47F F87A M238K V281G M354S D363H W575C A595T). P450 BM3 M9 was used for the continuous conversion of 3-phenoxytoluene in a plug flow reactor (PFR) since P450 BM3 M9 has a 3-fold higher activity for 3-phenoxytoluene compared to P450 BM3 M7 which was used for optimizing immobilization conditions with the highest activity for 12-pNCA assay. The reactor could be operated for 5 days with total turnover numbers (TTNs) over 2,000. [ABSTRACT FROM AUTHOR]

Cytochrome P450 cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450 cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate. Mutant (E156G/V247F/V253G/F256S) had the highest maximal velocity in the conversion of 3-chloroindole to isatin, whereas mutants (T56A/N116H/D297N) and (G60S/Y75H) had highest k cat / K M values. Six of the mutants had more than one mutation, and within this set, mutation of residues 297 and 179 was observed twice. Docking simulations were performed on models of the mutant enzymes; the wild-type did not accommodate 3-chloroindole in the active site, whereas all the mutants did. We propose two potential reaction pathways for dechlorination of 3-chloroindole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. [ABSTRACT FROM AUTHOR]

Biocatalysis with cytochrome P450 enzymes are important for the industrial production of fine chemicals, pharmaceuticals, fragrance and flavor compounds since chemoselective hydroxylation of aromatics and terpenes are chemically difficult to achieve. A few P450 based industrial processes have been developed based on whole cell catalysis. However, the outer membrane of microbial cells forms an effective barrier, which reduces the uptake of hydrophobic substrates. The coexpression of outer membrane proteins in E. coli such as the ferric hydroxamate uptake protein (FhuA) can provide alternative solutions to chemical or physical methods for increasing compound flux through the outer membrane of E. coli and thereby to boost productivities. In this study we employed an engineered FhuA Δ1-160 variant in which the “cork domain” was removed (first 160 residues are deleted); FhuA Δ1-160 has a cross-section of 39–46 Å with a “free” inner diameter of about 14 Å that serves as passive diffusion channel. FhuA WT and Δ1-160 were coexpressed on a bicistronic system with two P450 BM3 variants for regiospecific hydroxylation of aromatic compounds toluene and anisole as well as for oxidation of two terpenes (α)-pinene and ( R )-(+)-limonene. The presence of FhuA Δ1‐160 resulted in a doubled product concentration for toluene (35 μ to 50 μM), anisole (25 μM to 45 μM), pinene (12 μM to 20 μM) and limonene (12 μM to 25 μM) and five times higher for the coumarin derivative BCCE. In order to characterizes and compensate for expression variations a quantification method based on Chromeo546-labled StrepTactinII was developed to quantify the number of FhuA Δ1-160 in the outer E. coli membrane (∼44000 of FhuA Δ1-160 per cell). Morphology studies showed that a 6% E. coli surface coverage can be achieved with FhuA Δ1‐160 without significantly influencing the E. coli rod shape. In summary, FhuA Δ1-160 efficiently increases uptake of hydrophobic aromatics and terpenes for whole-cell biotransformations and can likely be used for other enzymes and/or substrates. [ABSTRACT FROM AUTHOR]

Abstract: P450s catalyze a wide spectrum of stereo- and regioselective reactions like hydroxylations, epoxidations and dehydrogenations. Therefore biotransformations with P450s are of great relevance to organic synthesis. The use of isolated enzymes offers advantages over the use of whole cells. A key issue for catalytic applications of isolated P450s is the demand for a continuous electron supply to the heme-group. Mediator driven bioelectrocatalysis can help to overcome this problem. For mediator driven bioelectrocatalysis the identification of a suitable mediator is crucial for the fast development of an efficient electro enzymatic process. To this end we have developed a computational screening method based on using freely available software. Calculated electron transfer rates were compared with measured product formation rates. The novel in silico procedure allows a faster identification of suitable mediators for electrochemically driven P450 catalyzed reactions and can be used as screening tool. It may also lead to a massive reduction of experimental effort for the development of bioelectrochemical reaction systems in the future. [Copyright &y& Elsevier]

Abstract: P450cin stereoselectively hydroxylated its natural substrate 1,8-cineole to 2β-hydroxy-1,8-cineole in an electrochemical cell which allowed for substitution of the natural cofactor NADPH by artificial redox mediators. Cobalt sepulchrate, phenosafranine, safranine T, FAD and FMN enabled artificial electron transfer from the platinum electrode to P450cin via the redox partner protein cindoxin. The highest product formation, 6.50±0.60nmol (product) nmol (P450)−1 min−1 cm−2, was achieved using cobalt sepulchrate. Surprisingly, phenosafranine and safranine T enabled electron transfer even in the absence of NADPH, cindoxin, and cindoxin reductase, thereby illustrating that none of the natural redox partners is needed for product formation. [ABSTRACT FROM AUTHOR]