Cytochrome P450 BM3 Monooxygenase Research Articles

Choose Your Own Adventure: A Comprehensive Database of Reactions Catalyzed by Cytochrome P450 BM3 Variants.

Cytochrome P450 BM3 monooxygenase is the topic of extensive research as many researchers have evolved this enzyme to generate a variety of products. However, the abundance of information on increasingly diversified variants of P450 BM3 that catalyze a broad array of chemistry is not in a format that enables easy extraction and interpretation. We present a database that categorizes variants by their catalyzed reactions and includes details about substrates to provide reaction context. This database of >1500 P450 BM3 variants is downloadable and machine-readable and includes instructions to maximize ease of gathering information. The database allows rapid identification of commonly reported substitutions, aiding researchers who are unfamiliar with the enzyme in identifying starting points for enzyme engineering. For those actively engaged in engineering P450 BM3, the database, along with this review, provides a powerful and user-friendly platform to understand, predict, and identify the attributes of P450 BM3 variants, encouraging the further engineering of this enzyme.

Enabling highly (R)-enantioselective epoxidation of styrene by engineering unique non-natural P450 peroxygenases.

Unlike the excellent (S)-enantioselective epoxidation of styrene performed by natural styrene monooxygenases (ee > 99%), the (R)-enantioselective epoxidation of styrene has not yet achieved a comparable efficiency using natural or engineered oxidative enzymes. This report describes the H2O2-dependent (R)-enantioselective epoxidation of unfunctionalized styrene and its derivatives by site-mutated variants of a unique non-natural P450BM3 peroxygenase, working in tandem with a dual-functional small molecule (DFSM). The observed (R)-enantiomeric excess (ee) of styrene epoxidation is up to 99% with a turnover number (TON) of 918 by the best enantioselective mutant F87A/T268I/L181Q, while the best active mutant F87A/T268I/V78A/A184L (with 98% ee) gave a catalytic TON of 4350, representing the best activity of a P450 peroxygenase towards styrene epoxidation to date. Following this approach, a set of styrene derivatives, such as o-, m-, p-chlorostyrenes and fluorostyrenes, could also be epoxidized with modest to very good TONs (362–3480) and high (R)-enantioselectivities (95–99% ee). The semi-preparative scale synthesis of (R)-styrene oxide performed at 0 °C with high conversion, maintaining enantioselectivity, and moderate isolated yields, further suggests the potential application of the current P450 enzymatic system in styrene epoxidation. This study indicates that the synergistic use of protein engineering and an exogenous DFSM constitutes an efficient strategy to control the enantioselectivity of styrene epoxidation, thus substantially expanding the chemical scope of P450 enzymes as useful bio-oxidative catalysts.

Chemo- and Regioselective Dihydroxylation of Benzene to Hydroquinone Enabled by Engineered Cytochrome P450 Monooxygenase.

Hydroquinone (HQ) is produced commercially from benzene by multi-step Hock-type processes with equivalent amounts of acetone as side-product. We describe an efficient biocatalytic alternative using the cytochrome P450-BM3 monooxygenase. Since the wildtype enzyme does not accept benzene, a semi-rational protein engineering strategy was developed. Highly active mutants were obtained which transform benzene in a one-pot sequence first into phenol and then regioselectively into HQ without any overoxidation. A computational study shows that the chemoselective oxidation of phenol by the P450-BM3 variant A82F/A328F leads to the regioselective formation of an epoxide intermediate at the C3=C4 double bond, which departs from the binding pocket and then undergoes fragmentation in aqueous medium with exclusive formation of HQ. As a practical application, an E. coli designer cell system was constructed, which enables the cascade transformation of benzene into the natural product arbutin, which has anti-inflammatory and anti-bacterial activities.

Selective Oxidations of Cyperenoic Acid by Slightly Reshaping the Binding Pocket of Cytochrome P450 BM3

AbstractThe selective hydroxylation of unactivated C−H bonds in complex natural products represents a formidable challenge in synthetic organic chemistry. Cyperenoic acid, a sesquiterpenoid isolated from Croton crassifolius, possesses an antiangiogenic‐promoting effect. Its C7‐ and C9α‐hydroxylated products can significantly inhibit the release of vascular endothelial growth factor (VEGF). To prepare these hydroxylated products, cytochrome P450 BM3 monooxygenase was chosen as a catalyst for the hydroxylation of cyperenoic acid. A simple and fast strategy, slightly reshaping the binding pocket of P450 BM3, was described to expedite the development of highly regio‐ and stereoselective P450 catalysts. P450 BM3 was evolved through one or two generations of mutations, and a highly enriched mutant library that contained fewer than 30 variants was then constructed. The obtained P450 BM3 variants achieved selective hydroxylation at positions C7 (94 % selectivity in the case of the F87A/A330W/F331L mutant) and C9 (90 % regioselectivity and 100 % stereoselectivity in the case of the L75V/F87A/T88F/A330W mutant) of cyperenoic acid and were also used to prepare the desired hydroxylated products at a preparative scale with high isolated yields.

Evaluation of coumarin-based fluorogenic P450 BM3 substrates and prospects for competitive inhibition screenings

Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458min−1μM−1). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP+ along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research.

The Mutagenesis Assistant Program.

Mutagenesis Assistant Program (MAP) is a web-based statistical tool to develop directed evolution strategies by investigating the consequences at the amino acid level of the mutational biases of random mutagenesis methods on any given gene. The latest development of the program, the MAP(2.0)3D server, correlates the generated amino acid substitution patterns of a specific random mutagenesis method to the sequence and structural information of the target protein. The combined information can be used to select an experimental strategy that improves the chances of obtaining functionally efficient and/or stable enzyme variants. Hence, the MAP(2.0)3D server facilitates the "in silico" prescreening of the target gene by predicting the amino acid diversity generated in a random mutagenesis library. Here, we describe the features of MAP(2.0)3D server by analyzing, as an example, the cytochrome P450BM3 monooxygenase (CYP102A1). The MAP(2.0)3D server is available publicly at http://map.jacobs-university.de/map3d.html.

Benzylic hydroxylation of aromatic compounds by P450 BM3

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.

The role of active-site Phe87 in modulating the organic co-solvent tolerance of cytochrome P450 BM3 monooxygenase.

Understanding the effects of organic co-solvents on protein structure and function is pivotal to engineering enzymes for biotransformation in non-aqueous solvents. The effects of DMSO on the catalytic activity of cytochrome P450 BM3 have previously been investigated and the importance of Phe87 in its organic co-solvent tolerance was identified. To probe the DMSO inactivation mechanism and the functional role of Phe87 in modulating the organic co-solvent tolerance of P450 BM3, the haem domain (Thr1-Leu455) of the F87A variant was cocrystallized in the presence of 14%(v/v) and 28%(v/v) DMSO. At both DMSO concentrations the protein retained the canonical structure of the P450 haem domain without any sign of partial or global unfolding. Interestingly, a DMSO molecule was found in the active site of both structures, with its O atom pointing towards the haem iron. The orientation of the DMSO molecule indicated a dynamic coordination process that was in competition with the active-site water molecule. The ability of the DMSO molecule to coordinate the haem iron is plausibly the main reason why P450 BM3 is inactivated at elevated DMSO concentrations. The data allowed an interesting comparison with the wild-type structures reported previously. A DMSO molecule was found when the wild-type protein was placed in 28%(v/v) DMSO, in which the DMSO molecule coordinated the haem iron directly via its S atom. Intriguingly, no DMSO molecule was observed at 14%(v/v) DMSO for the wild-type structure. These results suggested that the bulky phenyl side chain of Phe87 protects the haem from being accessed by the DMSO molecule and explains the higher tolerance of the wild-type enzyme towards organic co-solvents compared with its F87A variant.

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards γ- and δ-positions

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12–22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of ω-1, ω-2 and ω-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at δ-position (14% and 16%, respectively), but also produced γ- and β-hydroxylated products. δ-Hydroxy lauric and γ-hydroxy lauric acid are important synthons for the production of the corresponding lactones.

Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations.

Utilising whole cells of recombinant Escherichia coli K27 (pCYP102, pGEc47) containing active cytochrome P450BM-3 monooxygenase [E.C. 1. 14.14.1], multiple oxidations of saturated and unsaturated fatty acids were performed by the enzyme under conditions of excess oxygen. The amount of oxygen dissolved in the culture medium strongly influenced the regioselectivity of the reaction, as reflected in the distribution and amount of oxidised products. We have verified by gas chromatography/mass spectrometry that the products of in vivo biotransformation of pentadecanoic acid by cytochrome P450BM-3 are identical to those formed in cell-free extracts containing the enzyme. The formation of keto- and dihydroxy acids, side products which are characteristic for in vitro conversions with purified cytochrome P450BM-3 in the presence of excess oxygen, has been observed as well. Thus, by varying the oxygen concentration, we could control the regioselectivity of oxidation and the number of products made. Under oxygen limiting conditions, only monooxidised 12-, 13-, and 14-hydroxy-pentadecanoic acids were obtained. Consequently, unwanted side products could be excluded by modulating the amount of oxygen used in the bioconversion. Furthermore, whole cell oxidation of two unsaturated long-chain fatty acids, cis-pentadec-10-enoic and cis-hexadec-9-enoic acid, resulted in the production of epoxides, various subterminal hydroxyalkenoic acids and keto- and hydroxyalkanoic acids. Although we obtained higher activities of C15:0 conversion in vitro, the whole cell biocatalyst proved to be useful for specific oxidations of long-chain fatty acids since there is no need to add the costly cofactor NADPH. This biooxidation by E. coli K27 (pCYP102, pGEc47) under oxygen limitation has been demonstrated at the 2-L scale, showing that 12-, 13-, and 14-hydroxypentadecanoic acids can be produced in the g L-1 range.

Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM3 monooxygenase under varied dissolved oxygen concentrations

Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM3 monooxygenase under varied dissolved oxygen concentrations

Production Of Alkanedioic Acids By Cytochrome P450Bm-3 Monooxygenase: Oxidation Of 16-Hydroxyhexadecanoic Acid To Hexadecane-1, 16-Dioic Acid

Cytochrome P45BM-3 monooxygenase [E.C. 1.14.14.1] from Bacillus megaterium ATCC 14581 was used to devise a production route towards long chain alkanedioic acids. Both cell lysates and intact cells of an Escherichia coli JM101 recombinant, expressing the cyp102 gene that encodes cytochrome P450BM-3 monooxygenase, carried out the biotransformation of 16-hydroxyhexa-decanoic acid to 1, 16-hexadecanedioic acid. The intermediates of 16-hydroxyhexadecanoic acid oxidation by whole cells synthesizing cytochrome P450BM-3 monooxygenase were identified by gas chromatography mass spectrometry. Oxidation of 16-hydroxyhexadecanoic acid occurred with concomitant accumulation of small amounts of intermediates, one of which was tentatively identified as 16-dihydroxyhexadecanoic acid. Products of side reactions were 13.16-dihydroxy-hexadecanoic acid and 12, 16-dihydroxyhexadecanoic acid. The oxidation reactions did not take place in E. coli JM101 carrying the vector-DNA without the cyp102 gene.These data demonstrate for the first time that cytochrome P450BM-3 monooxygenase is able to oxidize ω-hydroxy long chain fatty acids to the corresponding dicarboxylic acids.

Production of chiral hydroxy long chain fatty acids by whole cell biocatalysis of pentadecanoic acid with an E. coli recombinant containing cytochrome P450 BM-3 monooxygenase

A multi-step product recovery process for 12-, 13- and 14-hydroxypentadecanoic acids was developed. Biotransformation of pentadecanoic acid by whole cells of recombinant E. coli K27 (pCYP102, pGEc47), synthesizing cytochrome P450 BM-3 monooxygenase [EC 1.14.14.1], led to the production of 12-, 13- and 14-hydroxypentadecanoic acids. To prevent further oxidation of these products, the conversion was performed under oxygen-limited conditions. After filtration and hexane extraction of culture supernatants, the hydroxy fatty acids were methylated. The purification process allowed us to separate the mixture of 12-, 13- and 14-hydroxypentadecanoic acid methyl esters into their single regioisomers by reversed-phase HPLC, followed by precipitation of the compounds as white crystals at 4°C and −20°C. The purified hydroxymethyl esters were characterized by GC–MS analysis. Chiral HPLC analysis of the ω-1 product, 14-hydroxypentadecanoic acid methyl ester, indicated that pentadecanoic acid is oxidized by cytochrome P450 BM-3 to optically pure ( S)-(+)-14-hydroxypentadecanoic acid at an e.e. of over 95%. This result confirms recent observations by Capdevila [1]that oxidation by cytochrome P450 BM-3 monooxygenase is highly stereoselective.

Alkane metabolism by cytochrome P450 BM3.

The cytochrome P450 BM3 monooxygenase system from Bncillus megnrerium provides a unique model system for mammalian class 11 P450s. including those involved in human drug metabolism. It is a flavocytochrome, containing both P450 and NADPH-P450 reductase redox partner in a single polypeptide chain, and is the only prokaryotic class I1 P450. It is also soluble, unlike its eukaryotic membranous counterparts. which greatly simplifies preparation and analysis (1.2).