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

Abstract

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.

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

“…BM3 is a soluble and stable fusion between a catalytic domain involved in substrate oxidation and a diflavin reductase domain responsible for electron transport, thereby making the electron transfer very efficient. These fine properties, together with the ease of over-expression in Escherichia coli ( E. coli ) made BM3 a promising candidate for the biocatalysis 3 , 4 , 5 . Therefore, BM3 was studied extensively and many BM3 mutants with broader substrate range and altered region- and stereo-selectivities were generated by laboratory evolution 6 , 7 , 8 .…”

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

“…All positions of the P450 BM3 heme domain that have been mutated and show improvement toward drug/drug-like hydroxylation are shown (orange spheres). Mutated positions in the heme domain (residues 1–471) and in the reductase domain (472–1049) are listed, with refs: 8 (), 11 ( , ), 23 ( , ), 24 ( , ), 39 (), 47 ( , , , , , , , , − , , , , , , , , , − ), 49 ( , , , , , ), 51 ( , , , , , , − , , , , , ), 52 ( , , ), 58 ( , , , ), 64 ( , , , , , , , , , …”

“…Screening a small number of variants with well-defined properties frequently results in the detection of variants with altered product oxygenation profiles. ,,,, Hydroxylation of testosterone by the R47L/F87V/L188Q variant generates three primary products, specifically 2β-, 15β-, and 16β-testosterone, whereas the wild-type is incapable of catalyzing this reaction . M01 and M11 are both active toward testosterone and show comparable product distributions, albeit M11 exhibits nearly 10 times higher activity than M01 .…”

“Commandeuring” Xenobiotic Metabolism: Advances in Understanding Xenobiotic Metabolism

“…An example of such an approach was shown with the screening of 83 individual mutants against a cocktail of six drugs. 107 It was established that the presence of multiple substrates did not significantly alter the percentage of substrate depletion nor the metabolic profile of the enzymes. Each of the 83 mutants was incubated individually with the cocktail of six drugs.…”

“…While the former method could be applied to study interactions between one mutant and one substrate, the screening process could be greatly enhanced using a cocktail approach. An example of such an approach was shown with the screening of 83 individual mutants against a cocktail of six drugs . It was established that the presence of multiple substrates did not significantly alter the percentage of substrate depletion nor the metabolic profile of the enzymes.…”