Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

Reinen, Jelle; Hemert, Daniel van; Vermeulen, Nico; Commandeur, Jan N. M.

doi:10.1177/1087057115607183

Cited by 9 publications

(13 citation statements)

References 29 publications

(31 reference statements)

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“…In another study, relative activity was studied, for several P450BM3 mutants built upon the w5f5 mutant, in the presence of increasing concentration of isopropanol, acetonitrile, DMSO, and methanol, with the latter two outperforming the other cosolvents. [15] This correlates with our own results (Table 1), where both methanol and DMSO display the highest enzyme melting temperatures of all organic cosolvents tested.…”

Section: Maximum Conversion Of 10-pnca To Pnp By P450bm3supporting

confidence: 91%

“…This can be alleviated by adding an organic cosolvent to the aqueous reaction phase, albeit at a low concentration to avoid enzyme deactivation. [13][14][15][16] As a result, the necessity for an organic cosolvent limits the stability and the product yield achievable by P450 enzymes and is therefore a principal hurdle to their industrial application. Unsurprisingly, the issue of cytochrome P450 stability has been repeatedly identified across reviews tackling the application of cytochrome P450 biotechnology as one of the main hurdles preventing their industrial implementation.…”

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confidence: 99%

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Optimization of operation conditions for improved cytochrome P450BM3 enzymatic reaction yield

2021

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The P450 cytochrome monooxygenase CYP102A1 from Bacillus megaterium, better known as P450BM3, is a heme‐thiolate enzyme that catalyzes the hydroxylation of numerous substrates. Many of the resulting products are of commercial interest to the pharmaceutical and fine chemical industries. Unlike most other P450 cytochromes, P450BM3 is both soluble and fused to its natural redox partner, which supplies the necessary electrons from NADPH to drive its reaction forward. However, the industrial use of this enzyme is limited by its poor stability and its expensive cofactor. In this work, we explore the effects of buffer formulation and temperature on the stability of wildtype and P450BM3 mutant R966D/W1046S as well as on the stability of nicotinamide cofactors NADPH, NADH, and the biomimetic cofactor N‐benzyl‐1,4‐dihydronicotinamide. We demonstrate that cofactor stability is more important to increase product yield than that of the enzyme. We also demonstrate that low temperatures enhance oxido‐reduction reactions coupling, thus resulting in an increase in the molar ratio of p‐nitrophenolate produced from 10‐pNCA per oxidized cofactor. Overall, the optimized reaction conditions lead to a 2 to 2.6‐fold increase in total product output when wildtype P450BM3 or R966D/W1046S mutant is used with either of these three aforementioned cofactors.

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Section: Maximum Conversion Of 10-pnca To Pnp By P450bm3supporting

confidence: 91%

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confidence: 99%

Optimization of operation conditions for improved cytochrome P450BM3 enzymatic reaction yield

2021

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“…Originally, the interaction of NpCN with HQ was described to occur in ethanol (Légrádi 1970 ). In contrast, P450s require aqueous systems for catalysis of hydroxylation reactions (Reinen et al 2015 ; Whitehouse et al 2012 ). The assay conditions were, thus, adjusted for the application in phosphate buffer (50 mM, pH 7.5) and downscaled into MTP format.…”

Section: Resultsmentioning

confidence: 99%

A hydroquinone-specific screening system for directed P450 evolution

Weingartner

Sauer

Dhoke

et al. 2018

Appl Microbiol Biotechnol

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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).Electronic supplementary materialThe online version of this article (10.1007/s00253-018-9328-3) contains supplementary material, which is available to authorized users.

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“…The 39 mutants that have not been described previously were all created in-house by site-directed mutagenesis using the appropriate mutant templates (see ESM, Table S2 ). Mutations have been introduced at different positions throughout the protein in order to address, amongst others, effects upon regio- and stereo-selectivity (residues 72, 74, 75, 81, 82, 86, 87, 264, 328, 436, 437 and 440), organic solvent tolerability (residues 235, 471, 494 and 1024, see also [ 36 ]) and protein stability (residues 53, 176, 208 and 359). In-depth discussion of these properties is not a subject in this manuscript and will not be addressed.…”

Section: Resultsmentioning

confidence: 99%

Application of a cocktail approach to screen cytochrome P450 BM3 libraries for metabolic activity and diversity

Reinen¹,

Postma

Tump

et al. 2016

Anal Bioanal Chem

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In the present study, the validity of using a cocktail screening method in combination with a chemometrical data mining approach to evaluate metabolic activity and diversity of drug-metabolizing bacterial Cytochrome P450 (CYP) BM3 mutants was investigated. In addition, the concept of utilizing an in-house-developed library of CYP BM3 mutants as a unique biocatalytic synthetic tool to support medicinal chemistry was evaluated. Metabolic efficiency of the mutant library towards a selection of CYP model substrates, being amitriptyline (AMI), buspirone (BUS), coumarine (COU), dextromethorphan (DEX), diclofenac (DIC) and norethisterone (NET), was investigated. First, metabolic activity of a selection of CYP BM3 mutants was screened against AMI and BUS. Subsequently, for a single CYP BM3 mutant, the effect of co-administration of multiple drugs on the metabolic activity and diversity towards AMI and BUS was investigated. Finally, a cocktail of AMI, BUS, COU, DEX, DIC and NET was screened against the whole in-house CYP BM3 library. Different validated quantitative and qualitative (U)HPLC-MS/MS-based analytical methods were applied to screen for substrate depletion and targeted product formation, followed by a more in-depth screen for metabolic diversity. A chemometrical approach was used to mine all data to search for unique metabolic properties of the mutants and allow classification of the mutants. The latter would open the possibility of obtaining a more in-depth mechanistic understanding of the metabolites. The presented method is the first MS-based method to screen CYP BM3 mutant libraries for diversity in combination with a chemometrical approach to interpret results and visualize differences between the tested mutants.Graphical abstractGeneral worklfow in screening mutant enzyme libraries for catalytic efficiency and diversityElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-015-9241-x) contains supplementary material, which is available to authorized users.

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Application of a Continuous-Flow Bioassay to Investigate the Organic Solvent Tolerability of Cytochrome P450 BM3 Mutants

Cited by 9 publications

References 29 publications

Optimization of operation conditions for improved cytochrome P450BM3 enzymatic reaction yield

Optimization of operation conditions for improved cytochrome P450BM3 enzymatic reaction yield

A hydroquinone-specific screening system for directed P450 evolution

Application of a cocktail approach to screen cytochrome P450 BM3 libraries for metabolic activity and diversity

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