Fungal Cytochrome P450 Research Articles

A broad-range yeast expression system revealsArxula adeninivoransexpressing a fungal self-sufficient cytochrome P450 monooxygenase as an excellent whole-cell biocatalyst

The feasibility of using a single vector to clone a cytochrome P450 monooxygenase (P450) in different yeasts and then compare whole-cell hydroxylase activity was investigated. A broad-range yeast expression vector using the ylTEFp to drive expression of the cloned gene and the scTEFp to drive the hygromycin resistance marker gene was used to clone the genes encoding two self-sufficient P450s, CYP102A1 and CYP505A1. Both genes were cloned into Saccharomyces cerevisiae, Kluyveromyces marxianus, Yarrowia lipolytica (two strains) and Arxula adeninivorans. 4-Hexylbenzoic acid (HBA), which is subterminally hydroxylated by both CYP102A1 and CYP505A1, was used to compare whole-cell hydroxylase activity of transformants. Kluyveromyces marxianus and A.adeninivorans exhibited activity with both CYP102A1 and CYP505A1, while S.cerevisiae only displayed CYP102A1 activity and Y.lipolytica only CYP505A1 activity. The highest CYP102A1 activity (0.8mM HBA converted in 24h) was observed with concentrated resting-cell suspensions of S.cerevisiae. The CYP505A1 activity observed with growing cultures of A.adeninivorans was however at least 12 times higher than the CYP102A1 activity of S.cerevisiae with up to 2mM HBA converted within 6h. The use of K.marxianus and A.adeninivorans for P450 expression has not previously been reported.

Methods for efficient high-throughput screening of protein expression in recombinant Pichia pastoris strains.

The methylotrophic yeast Pichia pastoris is becoming one of the favorite industrial workhorses for protein expression. Due to the widespread use of integration vectors, which generates significant clonal variability, screening methods allowing assaying hundreds of individual clones are of particular importance. Here we describe methods to detect and analyze protein expression, developed in a 96-well format for high-throughput screening of recombinant P. pastoris strains. The chapter covers essentially three common scenarios: (1) an enzymatic assay for proteins expressed in the cell cytoplasm, requiring cell lysis; (2) a whole-cell assay for a fungal cytochrome P450; and (3) a nonenzymatic assay for detection and quantification of tagged protein secreted into the supernatant.

Synthesis, Antifungal Evaluation and Molecular Docking Studies on 2- thioxoimidazolidin-4-one Derivatives

A series of 2-thioxoimidazolidin-4-one derivatives were synthesized and were characterized by IR, 1H NMR, 13C NMR, mass spectral data and elemental analysis. All compounds were tested for antifungal and antibacterial activity. Some of the compounds were found to exhibit promising antifungal activity. Molecular modeling investigations showed that the active compounds may interact at the active site of the fungal cytochrome P450- dependent sterol 14α- demethylase in the sterol biosynthesis pathway.

Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction.

Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts via attachment to cuticular substrata and the production of enzymes for the degradation and penetration of insect cuticle. The outermost epicuticular layer consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain hydrocarbons are major components of the outer waxy layer of diverse insect species, where they serve to protect against desiccation and microbial parasites, and as recognition molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with overlapping substrate specificities. Alkanes and fatty acids are substrates for a specific subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon degradation. These enzymes activate alkanes by terminal oxidation to alcohols, which are further oxidized by alcohol and aldehyde dehydrogenases, whose products can enter β-oxidation pathways. B. bassiana contains at least 83 genes coding for cytochrome P450s (CYP), a subset of which are involved in hydrocarbon oxidation, and several of which represent new CYP subfamilies/families. Expression data indicated differential induction by alkanes and insect lipids and four CYP proteins have been partially characterized after heterologous expression in yeast. Gene knockouts revealed a phenotype for only one (cyp52X1) out of six genes examined to date. CYP52X1 oxidizes long chain fatty acids and participates in the degradation of specific epicuticular lipid components needed for breaching the insect waxy layer. Examining the hydrocarbon oxidizing CYP repertoire of pathogens involved in insect epicuticle degradation can lead to the characterization of enzymes with novel substrate specificities. Pathogen targeting may also represent an important co-evolutionary process regarding insect cuticular hydrocarbon synthesis.

Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes

BackgroundCytochrome P450 proteins (CYPs) play diverse and pivotal roles in fungal metabolism and adaptation to specific ecological niches. Fungal genomes encode extremely variable “CYPomes” ranging from one to more than 300 CYPs. Despite the rapid growth of sequenced fungal and oomycete genomes and the resulting influx of predicted CYPs, the vast majority of CYPs remain functionally uncharacterized. To facilitate the curation and functional and evolutionary studies of CYPs, we previously developed Fungal Cytochrome P450 Database (FCPD), which included CYPs from 70 fungal and oomycete species. Here we present a new version of FCPD (1.2) with more data and an improved classification scheme.ResultsThe new database contains 22,940 CYPs from 213 species divided into 2,579 clusters and 115 clans. By optimizing the clustering pipeline, we were able to uncover 36 novel clans and to assign 153 orphan CYP families to specific clans. To augment their functional annotation, CYP clusters were mapped to David Nelson’s P450 databases, which archive a total of 12,500 manually curated CYPs. Additionally, over 150 clusters were functionally classified based on sequence similarity to experimentally characterized CYPs. Comparative analysis of fungal and oomycete CYPomes revealed cases of both extreme expansion and contraction. The most dramatic expansions in fungi were observed in clans CYP58 and CYP68 (Pezizomycotina), clans CYP5150 and CYP63 (Agaricomycotina), and family CYP509 (Mucoromycotina). Although much of the extraordinary diversity of the pan-fungal CYPome can be attributed to gene duplication and adaptive divergence, our analysis also suggests a few potential horizontal gene transfer events. Updated families and clans can be accessed through the new version of the FCPD database.ConclusionsFCPD version 1.2 provides a systematic and searchable catalogue of 9,550 fungal CYP sequences (292 families) encoded by 108 fungal species and 147 CYP sequences (9 families) encoded by five oomycete species. In comparison to the first version, it offers a more comprehensive clan classification, is fully compatible with Nelson’s P450 databases, and has expanded functional categorization. These features will facilitate functional annotation and classification of CYPs encoded by newly sequenced fungal and oomycete genomes. Additionally, the classification system will aid in studying the roles of CYPs in the evolution of fungal adaptation to specific ecological niches.

Fungal cytochrome P450 sterol 14α-demethylase (CYP51) and azole resistance in plant and human pathogens

Azoles have been applied widely to combat pathogenic fungi in medicine and agriculture and, consequently, loss of efficacy has occurred in populations of some species. Often, but not always, resistance was found to result from amino acid substitutions in the molecular target of azoles, 14α-sterol demethylase (CYP51 syn. ERG11). This review summarizes CYP51 function, evolution, and structure. Furthermore, we compare the occurrence and contribution of CYP51 substitutions to azole resistance in clinical and field isolates of important fungal pathogens. Although no crystal structure is available yet for any fungal CYP51, homology modeling using structures from other origins as template allowed deducing models for fungal orthologs. These models served to map amino acid changes known from clinical and field isolates. We conclude with describing the potential consequences of these changes on the topology of the protein to explain CYP51-based azole resistance. Knowledge gained from molecular modeling and resistance research will help to develop novel azole structures.

Functional Expression and Characterization of Recombinant NADPHP450 Reductase from Malassezia globosa

Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

Heterologous expression and mechanistic investigation of a fungal cytochrome P450 (CYP5150A2): Involvement of alternative redox partners

A fungal cytochrome P450 monooxygenase (CYP5150A2) from the white-rot basidiomycete Phanerochaete chrysosporium was heterologously expressed in Escherichia coli and purified as an active form. The purified CYP5150A2 was capable of hydroxylating 4-propylbenzoic acid (PBA) with NADPH-dependent cytochrome P450 oxidoreductase (CPR) as the single redox partner; the reaction efficiency was improved by the addition of electron transfer protein cytochrome b5 (Cyt-b5). Furthermore, CYP5150A2 exhibited substantial activity with redox partners Cyt-b5 and NADH-dependent Cyt-b5 reductase (CB5R) even in the absence of CPR. These results indicated that a combination of CB5R and Cyt-b5 may be capable of donating both the first and the second electrons required for the monooxygenation reaction. Under reaction conditions in which the redox system was associated with the CB5R-dependent Cyt-b5 reduction system, the exogenous addition of CPR and NADPH had no effect on the PBA hydroxylation rate or on coupling efficiency, indicating that the transfer of the second electron from Cyt-b5 was the rate-limiting step in the monooxygenase system. In addition, the rate of PBA hydroxylation was significantly dependent on Cyt-b5 concentration, exhibiting Michaelis–Menten kinetics. This study provides indubitable evidence that the combination of CB5R and Cyt-b5 is an alternative redox partner facilitating the monooxygenase reaction catalyzed by CYP5150A2.

Electrochemical investigations of fungal cytochrome P450

The electrochemical investigations of fungal ( Aspergillus terreus MTCC 6324) cytochrome P450 monooxygenase (CYP) (EC 1.14.14.1) immobilized on multi-walled carbon nanotubes–Nafion®–polyethyleneimine (MWCNT-NF/PEI) modified glassy carbon electrode (GCE) were carried out to understand the electrochemistry of the CYP and its application potential in electrochemical-based devices. In deoxygenated condition, the formal potential for Fe (III)/Fe (II) redox couple of CYP was about −0.53 V (vs. Ag/AgCl reference electrode, pH 8). A positive shift in the redox potential of the bioelectrode to −0.475 V was observed in the presence of oxygen and substrate. The electrocatalytic activity towards n-hexadecane and inhibition with piperonyl butoxide (PBO) confirmed the involvement of Fe (III/II) system of CYP in the redox process. The current response increased linearly from 1 μM to 100 μM of n-hexadecane with detection limit 0.1 μM. The IC 50 for PBO was 2.7 μM. The surface coverage of CYP immobilized on MWCNT-NF/PEI modified GCE was approximately 3.45 × 10 −10 mol cm −2. The electron transfer rate constant ( k s ) was 1.0 ± 0.2 s −1, indicating facilitation of the electron transfer between CYP and GCE.

The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification

Cytochromes P450 (CYPs) catalyse diverse reactions and are key enzymes in fungal primary and secondary metabolism, and xenobiotic detoxification. CYP enzymatic properties and substrate specificity determine the reaction outcome. However, CYP-mediated reactions may also be influenced by their redox partners. Filamentous fungi with numerous CYPs often possess multiple microsomal redox partners, cytochrome P450 reductases (CPRs). In the plant pathogenic ascomycete Cochliobolus lunatus we recently identified two CPR paralogues, CPR1 and CPR2. Our objective was to functionally characterize two endogenous fungal cytochrome P450 systems and elucidate the putative physiological roles of CPR1 and CPR2. We reconstituted both CPRs with CYP53A15, or benzoate 4-hydroxylase from C. lunatus, which is crucial in the detoxification of phenolic plant defence compounds. Biochemical characterization using RP-HPLC shows that both redox partners support CYP activity, but with different product specificities. When reconstituted with CPR1, CYP53A15 converts benzoic acid to 4-hydroxybenzoic acid, and 3-methoxybenzoic acid to 3-hydroxybenzoic acid. However, when the redox partner is CPR2, both substrates are converted to 3,4-dihydroxybenzoic acid. Deletion mutants and gene expression in mycelia grown on media with inhibitors indicate that CPR1 is important in primary metabolism, whereas CPR2 plays a role in xenobiotic detoxification.

Biotransformation of the organochlorine pesticide trans-chlordane by wood-rot fungi

There is very limited information on the biotransformation of organochlorine pesticide chlordane by microorganisms, and no systematic study on the metabolic products and pathways for chlordane transformation by wood-rot fungi has been conducted. In this study, trans-chlordane was metabolized with the wood-rot fungi species Phlebia lindtneri, Phlebia brevispora and Phlebia aurea, which are capable of degrading polychlorinated dibenzo-p-dioxin and heptachlor epoxide. At the end of 42 days of incubation, over 50% of trans-chlordane was degraded by the fungal treatments in pure cultures. These fungi transformed trans-chlordane to at least eleven metabolites including a large amount of hydroxylated products such as 3-hydroxychlordane, chlordene chlorohydrin, heptachlor diol, monohydroxychlordene and dihydroxychlordene. P. lindtneri particularly can metabolize oxychlordane, a recalcitrant epoxide product of chlordane, into a hydroxylated product through substitution of chlorine atom by hydroxyl group. The present results suggest that hydroxylation reactions play an important role in the metabolism of trans-chlordane by these Phlebia species. Additionally, transformation of trans-chlordane and production of hydroxylated metabolites were efficiently inhibited by the addition of cytochrome P450 inhibitors, piperonyl butoxide and 1-aminobenzotriazole, demonstrating that fungal cytochrome P450 enzymes are involved in some steps of trans-chlordane metabolism, particularly in the hydroxylation process.

A Diversified Library of Bacterial and Fungal Bifunctional Cytochrome P450 Enzymes for Drug Metabolite Synthesis

AbstractInnovative biohydroxylation catalysts for the preparation of drug metabolites were developed from scratch. A set of bacterial and fungal sequences of putative and already known bifunctional P450 enzymes was identified by protein sequence alignments, expressed in Escherichia coli and characterised. Notably, a fungal self‐sufficient cytochrome P450 (CYP) from Aspergillus fumigatus turned out to be especially stable during catalyst preparation and application and also in presence of organic co‐solvents. To enhance the catalytic activity and broaden the substrate specificity of those variants with high expression levels prominent single mutations were introduced. Selected improved variants were then used as lyophilised bacterial lysates for the synthesis of 4′‐hydroxydiclofenac and 6‐hydroxychlorzoxazone, the two metabolites of active pharmaceutical compounds diclofenac and chlorzoxazone representing the same metabolites as generated by human P450s.

Prednisone reduces ketoconazole-induced skeletal defects in rat fetuses

Ketoconazole (KT) is a broad-spectrum antifungal agent whose pharmacological activity is based on the capability to interfere with steroid biosynthesis through an interaction with fungal cytochrome P-450 enzymes and thereby avoiding the formation of fungal walls. As the inhibition of fungal cytochrome P-450 by KT is not specific, the mammalian cytochrome P-450 species, which play an important role in the biosynthesis of steroidogenesis, are also affected. The reproductive and developmental toxicity of KT have been assessed. This antimycotic agent has been reported as embryotoxic and teratogenic when administered in high doses (80 mg/kg) to pregnant rats. The mechanisms by which KT exert teratogenic effects remains to be elucidated. When considering the potential inhibitory effect of KT on mammalian steroid biosynthesis as a possible responsible for the skeletal anomalies induced by this drug, this study aimed at determining whether steroid maternal supplementation may prevent the skeletal anomalies induced by KT. To test this hypothesis, maternal supplementation with prednisone (PRED) (0.1, 0.2 or 0.4 mg/kg) and 80 mg/kg of KT were administered to pregnant Wistar rats (n = 10) during organogenesis period. On gestational day 21, the dams were euthanized and examined for standard parameters of reproductive outcome. In summary, the results showed that PRED supplementation therapy may cause reductions in the incidence of KT-induced cranial and appendicular skeletal anomalies as well as cleft palate in the rat, being these results more consistent with 0.4 mg/kg of this drug. These results suggest an important role for glucocorticoids in KT-induced teratogenesis.

Functional Identification of a Riceent-Kaurene Oxidase, OsKO2, Using thePichia pastorisExpression System

Rice ent-kaurene oxidase 2 (OsKO2) perhaps functions in the early steps of gibberellin biosynthesis. We found that microsomes from the methylotropic yeast Pichia pastoris expressing both OsKO2 and a fungal cytochrome P450 monooxygenase (P450) reductase converted ent-kaurene to ent-kaurenoic acid. This is direct evidence that OsKO2 is involved in the sequential oxidation of ent-kaurene to ent-kaurenoic acid in gibberellin biosynthesis in rice.

Fungal cytochrome P450 database

BackgroundCytochrome P450 enzymes play critical roles in fungal biology and ecology. To support studies on the roles and evolution of cytochrome P450 enzymes in fungi based on rapidly accumulating genome sequences from diverse fungal species, an efficient bioinformatics platform specialized for this super family of proteins is highly desirable.ResultsThe Fungal Cytochrome P450 Database (FCPD) archives genes encoding P450s in the genomes of 66 fungal and 4 oomycete species (4,538 in total) and supports analyses of their sequences, chromosomal distribution pattern, and evolutionary histories and relationships. The archived P450s were classified into 16 classes based on InterPro terms and clustered into 141 groups using tribe-MCL. The proportion of P450s in the total proteome and class distribution in individual species exhibited certain taxon-specific characteristics.ConclusionThe FCPD will facilitate systematic identification and multifaceted analyses of P450s at multiple taxon levels via the web. All data and functions are available at the web site .

Extensive Chemometric Investigations of the Multidrug Resistance in Strains of the Phytopathogenic Fungus Penicillium Digitatum

AbstractMultidrug resistance activities pEC50 of sensitive, resistant, and moderately resistant strains of the pathogenic fungus Penicillium digitatum against triflumizole, fenarimol, bitertanol, pyrifenox, cycloheximide, acriflavine, and 4‐nitroquinoline‐N‐oxide were studied by principal component and hierarchical cluster analyses. Genome descriptors for fungal cytochrome P450 sterol 14α‐demethylase and multidrug efflux pump PMR1 were generated and correlated with pEC50 by partial least squares. Toxicants were modeled at the PM3 level. Novel Activity–Structure Relationships (ASRs) were established to predict toxicant structural features from biological activities and to identify and classify the strains. New types of relationships to model and predict biological activities are Quantitative Genome–Activity Relationship (QGAR) and Quantitative Genome/Structure–Activity Relationship (QGSAR). QGAR for demethylation inhibitors had a reasonable regression [Q2=0.79, R2=0.81, Standard Error of Validation (SEV)=0.34] and was extended into QGSAR with improved statistics (Q2=0.85, R2=0.87, SEV=0.29). Conformers of toxicants with common hydrogen bonding and aromatic ring geometry indicate possible interactions with receptors such as cytochromes, efflux pumps, and regulatory proteins which activate fungal multidrug resistance.

A fungal cytochrome P450 is expressed during the interaction between the fungal pathogenHeterobasidion annosumsensu lato and conifer trees

The infection-related expression of a Heterobasidion annosum (Fr.) Bref. sensu lato (s.l.) putative cytochrome P450 gene (CPM2) was analysed with realtime quantitative PCR. CPM2 was highly expressed after 20 days of growth in bark of living spruce trees, and up-regulated by nitrogen starvation on artificial media. Infection of pine seedlings in the presence of high-carbon medium results in low expression levels of CPM2, thus indicating that starvation is the primary regulatory factor for induction of this gene. The predicted cpm2 protein contains 507 amino acids with an estimated molecular mass of 56.1 kDa, and display all conserved amino acids of the cytochrome P450 protein family. The protein has a high similarity to the ord1/ordA O-methylsterigmatocystin oxidoreductases from Aspergillus flavus/A. parasiticus, responsible for catalysing the final step in aflatoxin biosynthesis. Results indicate that cpm2 is potentially important for pathogenicity in H. annosum s.l.

High diversity and complex evolution of fungal cytochrome P450 reductase: Cytochrome P450 systems

Cytochrome P450 reductase (CPR) is the redox partner of P450 monooxygenases, involved in primary and secondary metabolism of eukaryotes. Two novel CPR genes, sharing 34% amino acid identity, were found in the filamentous ascomycete Cochliobolus lunatus. Fungal genomes were searched for putative CPR enzymes. Phylogenetic analysis suggests that multiple independent CPR duplication events occurred in fungi, whereas P450–CPR fusion occurred before the diversification of Dikarya and Zygomycota. Additionally, losses of methionine synthase reductase were found in certain fungal taxa; a truncated form of this enzyme was conserved in Pezizomycotina. In fungi, high numbers of cytochrome P450 enzymes, multiple CPRs, and P450–CPR fusion proteins were associated with filamentous growth. Evolution of multiple CPR-like oxidoreductases in filamentous fungi might have been driven by the complexity of biochemical functions necessitated by their growth form, as opposed to yeast.

Regulation and heterologous expression of P450 enzyme system components of the white rot fungus Phanerochaete chrysosporium

Phanerochaete chrysosporium is widely used as a model organism to understand the physiology, enzymology, and genetics of lignin degradation by white rot fungi and is known for its ability to metabolize and detoxify a wide range of environmental chemicals. Our pre-genomic efforts and the recent whole genome sequencing by the Joint Genome Institute of the US-DOE have revealed that this fungus carries a well-developed P450 enzyme system, consisting of multiple P450 monooxygenases and a common P450 oxidoreductase. The entire P450ome of this organism comprises of ∼150 cytochrome P450 monooxygenases, mostly arranged in gene clusters and classifiable into multigene families. Except for the structurally and functionally conserved fungal P450 families such as CYP51, CYP61, and CYP53, other P450 enzymes in this organism have largely unknown function and will require functional characterization. These new P450 enzymes may likely have roles in biodegradation activity and physiology of this ligninolytic fungus. Our pre- and post-genomic efforts to understand the functional role of P450 enzyme systems in P. chrysosporium have focused on the regulation of expression of the first identified family of P450 enzymes, the CYP63 family, and genome-wide regulation of the other P450 families using a custom-designed P450 microarray. The genomically linked CYP63 member P450s were found to be differentially regulated under varying physiological and/or biodegradation conditions. Results on the heterologous expression of this family of monooxygenases in different prokaryotic and eukaryotic expression systems are presented and the inherent problems associated with the expression of these membrane proteins are discussed. Further, we report the expression and purification of the white rot fungal cytochrome P450 oxidoreductase (POR), the electron transfer component of its P450 enzyme system, required for P450 catalysis. The reported studies have uncovered the hitherto unknown regulatory aspects of the P450 enzyme system in P. chrysosporium and generated useful expression tools and knowledge base to pursue further studies on functional analysis of the P450 contingent in this model white rot fungus.

In Vitro Metabolism of the Fungicide and Environmental Contaminant trans-bromuconazole and Implications for Risk Assessment

trans-Bromuconazole is a chiral chemical representative of a class of triazole derivatives known to inhibit specific fungal cytochrome P-450 (CYP) reactions. Kinetic measurements and delineation of metabolic pathways for triazole chemicals within in vitro hepatic microsomes are needed for accurate risk assessment and predictive in vivo physiological modeling. The studies described here were conducted with rat liver microsomes to determine Michaelis–Menten saturation kinetic parameters (V max and K M) for trans-bromuconazole using both substrate depletion and product formation reaction velocities. Kinetic parameters determined for trans-bromuconazole depletion at varying protein levels incubated at physiological temperature 37°C resulted in a K M value of 1.69 μM and a V max value of 1398 pmol/min/mg protein. The concomitant linear formation of two metabolites identified using liquid chromatography/time-of-flight mass spectrometry (LC/MS-TOF) and LC-MS/MS indicated hydroxylation of the trans-bromuconazole dichlorophenyl ring moiety. K M values determined for the hydroxylated metabolites were 0.87 and 1.03 μM, with V max values of 449 and 694 pmol/min/mg protein, respectively. Chemical inhibition assays and studies conducted with individual purified human recombinant enzymes indicated the CYP3A subfamily was primarily responsible for biotransformation of the parent substrate. Additionally, trans-bromuconazole was found to undergo stereoselective metabolism as evidenced by a change in the enantiomeric ratio (trans−/trans +) with respect to time.