Application of cytochrome P450 BM3 mutants as biocatalysts for the profiling of estrogen receptor binding metabolites of the mycotoxin zearalenone

Abstract

The estrogenic mycotoxin zearalenone (ZEN) can undergo hepatic reductive metabolism to form the estrogenic α and β isomers of zearalenol. ZEN also undergoes cytochrome P450 monooxygenase (P450)-mediated oxidative metabolism to form monohydroxylated products, but until now nothing is known about the estrogenic potency of these metabolites. This study aimed at investigating the metabolism of ZEN by different P450 isoforms and to determine the estrogen receptor α (ERα) affinities of the in vitro P450-generated ZEN metabolites in an online high-resolution screening (HRS) setup. Human liver microsomes (HLM), recombinant P450s, and mutants of the bacterial P450 BM3 were used to investigate the oxidative metabolism of ZEN. It was shown that mutants of the bacterial P450 BM3 could be used to produce the human relevant 13- and 15-OH-ZEN catechol metabolites at such levels that their ERα affinity could be determined in an HRS setup, which was not possible with HLM. It was demonstrated that P450-mediated hydroxylation at the 13 and 15 positions of ZEN resulted in a loss of ERα affinity. The approach presented here can be used for the elucidation of the metabolism of other endocrine disrupting compounds and xenobiotics to get clear pictures of the total effects of these compounds and their metabolites.

Combination of LC-MS2 and GC-MS as a Tool to Differentiate Oxidative Metabolites of Zearalenone with Different Chemical Structures

“…As an example, the MS 2 information provided in this paper is applied to the recent report by Reinen et al [10] on the ZEN metabolites formed by bacterial cytochrome P450 mutants. Six oxidative metabolites, designated M1, M2, M3A, M3B, M4, and M5, were detected and the MS 2 , obtained by LC-MS 2 with a 3D ion trap mass spectrometer operated with negative APCI, depicted in their paper [10]. M3A and M3B were tentatively proposed to represent products of aromatic hydroxylation, that is, at C-13 and C-15, and no structures were proposed for the remaining metabolites other than hydroxylation at the aliphatic macrocycle.…”

Selective whole-cell biosynthesis of the designer drug metabolites 15- or 16-betahydroxynorethisterone by engineered Cytochrome P450 BM3 mutants

“…The 40 other previously described mutants have been based upon these four templates [28][29][30][31][32][33][34]. The 44 mutants have been shown to metabolize a wide range of substrates, including marketed drugs [28,30,35,36], steroids [29,32,34], ionones [33], kinase inhibitors [47], alkoxyresorufins [29,31], and endocrine disrupting chemicals [31,48], with varying catalytic activities while also displaying significant differences in the metabolic profiles generated. The 22 mutants that have not been described before were all created inhouse by site-directed mutagenesis using the appropriate mutant templates (see the Supporting Information, Table S2).…”

“…Also within our own lab, isolated CYP BM3 mutants have previously been successfully applied for biosynthesis of drug metabolites on a preparative scale [32][33][34][35][36]39,47,48]. During our in vitro experiment, bioconversion was monitored in time (see Fig.…”

Directed evolution of cytochrome P450 enzymes for biocatalysis: exploiting the catalytic versatility of enzymes with relaxed substrate specificity

“…An exception is the evolution of P450 BM3 and P450cam mutants for activity towards various alkanes and terpenes (for examples, see [86][87][88][89][90][91]). Nonetheless, assays for enantioselectivity [93] and production of biologically active metabolites [94] have been designed in which the activity or property of interest can be monitored by a simple colorimetric or fluorimetric assay. However, few products of industrial relevance can be detected by facile colorimetric or fluorimetric methods.…”

“…In a pioneering study, the generation of oestrogenic metabolites of zearalenone by P450 102 mutants was coupled to the inline detection of affinity for the oestrogen receptor (ER) [94]. Conceivably, extracts from incubations of a lead compound with a targeted library of P450s could be assayed directly for the desired pharmacological activity.…”