The Dark Side of the pollen: BSA-seq identified genomic regions linked to male sterility in globe artichoke.

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke's significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

Ultrasound-Driven Deposition of Au and Ag Nanoparticles on Citrus Pectin: Preparation and Characterisation of Antimicrobial Composites.

Pectin is a renewable, non-toxic and biodegradable polymer made of galacturonic acid units. Its polar groups make it suitable for complexing and supporting metallic nanoparticles (NPs). This work aimed to produce antibacterial nanocomposites using pectin and acoustic cavitation. The metal NPs (Au or Ag) were deposited using ultrasound (US, 21 kHz, 50 W) and compared with those achieved with mechanical stirring. The impact of the reducing agents (NaBH4, ascorbic acid) on the dispersion and morphology of the resulting NPs was also assessed. Characterization by diffuse reflectance (DR) UV-Vis-NIR spectroscopy and field emission scanning electron microscopy (FESEM) showed that the use of US improves the dispersion and decreases the size of both Au and Ag NPs. Moreover, with Au NPs, avoiding external reductants led to smaller NPs and more uniform in size. The prepared NPs were functionalized with oxytetracycline in water and tested against Escherichia coli (gram negative) and Staphylococcus epidermidis (gram positive) via the Kirby-Bauer test. The results show a better antibacterial activity of the functionalized nanoparticles compared to antibiotic-free NPs and pure oxytetracycline, advising the potential of the nanoparticles as drug carriers. These findings underscore the significance of US-assisted synthesis, paving the way to new environmentally friendly antimicrobial materials.

Heme Spin Distribution in the Substrate-Free and Inhibited Novel CYP116B5hd: A Multifrequency Hyperfine Sublevel Correlation (HYSCORE) Study.

The cytochrome P450 family consists of ubiquitous monooxygenases with the potential to perform a wide variety of catalytic applications. Among the members of this family, CYP116B5hd shows a very prominent resistance to peracid damage, a property that makes it a promising tool for fine chemical synthesis using the peroxide shunt. In this meticulous study, we use hyperfine spectroscopy with a multifrequency approach (X- and Q-band) to characterize in detail the electronic structure of the heme iron of CYP116B5hd in the resting state, which provides structural details about its active site. The hyperfine dipole-dipole interaction between the electron and proton nuclear spins allows for the locating of two different protons from the coordinated water and a beta proton from the cysteine axial ligand of heme iron with respect to the magnetic axes centered on the iron. Additionally, since new anti-cancer therapies target the inhibition of P450s, here we use the CYP116B5hd system-imidazole as a model for studying cytochrome P450 inhibition by an azo compound. The effects of the inhibition of protein by imidazole in the active-site geometry and electron spin distribution are presented. The binding of imidazole to CYP116B5hd results in an imidazole-nitrogen axial coordination and a low-spin heme FeIII. HYSCORE experiments were used to detect the hyperfine interactions. The combined interpretation of the gyromagnetic tensor and the hyperfine and quadrupole tensors of magnetic nuclei coupled to the iron electron spin allowed us to obtain a precise picture of the active-site geometry, including the orientation of the semi-occupied orbitals and magnetic axes, which coincide with the porphyrin N-Fe-N axes. The electronic structure of the iron does not seem to be affected by imidazole binding. Two different possible coordination geometries of the axial imidazole were observed. The angles between gx (coinciding with one of the N-Fe-N axes) and the projection of the imidazole plane on the heme were determined to be -60° and -25° for each of the two possibilities via measurement of the hyperfine structure of the axially coordinated 14N.

Posttranslational Modification of Human Cytochrome CYP4F11 by 4-Hydroxynonenal Impairs ω-Hydroxylation in Malaria Pigment Hemozoin-Fed Monocytes: The Role in Malaria Immunosuppression.

Malaria is a frequent parasitic infection becomes life threatening due to the disequilibrated immune responses of the host. Avid phagocytosis of malarial pigment hemozoin (HZ) and HZ-containing Plasmodium parasites incapacitates monocyte functions by bioactive lipoperoxidation products 4-hydroxynonenal (4-HNE) and hydroxyeicosatetraenoic acids (HETEs). CYP4F conjugation with 4-HNE is hypothesised to inhibit ω-hydroxylation of 15-HETE, leading to sustained monocyte dysfunction caused by 15-HETE accumulation. A combined immunochemical and mass-spectrometric approach identified 4-HNE-conjugated CYP4F11 in primary human HZ-laden and 4-HNE-treated monocytes. Six distinct 4-HNE-modified amino acid residues were revealed, of which C260 and H261 are localized in the substrate recognition site of CYP4F11. Functional consequences of enzyme modification were investigated on purified human CYP4F11. Palmitic acid, arachidonic acid, 12-HETE, and 15-HETE bound to unconjugated CYP4F11 with apparent dissociation constants of 52, 98, 38, and 73 µM, respectively, while in vitro conjugation with 4-HNE completely blocked substrate binding and enzymatic activity of CYP4F11. Gas chromatographic product profiles confirmed that unmodified CYP4F11 catalysed the ω-hydroxylation while 4-HNE-conjugated CYP4F11 did not. The 15-HETE dose dependently recapitulated the inhibition of the oxidative burst and dendritic cell differentiation by HZ. The inhibition of CYP4F11 by 4-HNE with consequent accumulation of 15-HETE is supposed to be a crucial step in immune suppression in monocytes and immune imbalance in malaria.

Catalytically self-sufficient CYP116B5: Domain switch for improved peroxygenase activity.

Self-sufficient cytochromes P450 of the sub-family CYP116B have gained great attention in biotechnology due to their ability to catalyze challenging reactions toward a wide range of organic compounds. However, these P450s are often unstable in solution and their activity is limited to a short reaction time. Previously it has been shown that the isolated heme domain of CYP116B5 can work as a peroxygenase with H2 O2 without the addition of NAD(P)H. In this work, protein engineering was used to generate a chimeric enzyme (CYP116B5-SOX), in which the native reductase domain is replaced by a monomeric sarcosine oxidase (MSOX) capable of producing H2 O2 . The full-length enzyme (CYP116B5-fl) is characterized for the first time, allowing a detailed comparison to the heme domain (CYP116B5-hd) and CYP116B5-SOX. The catalytic activity of the three forms of the enzyme was studied using p-nitrophenol as substrate, and adding NADPH (CYP116B5-fl), H2 O2 (CYP116B5-hd), and sarcosine (CYP116B5-SOX) as source of electrons. CYP116B5-SOX performs better than CYP116B5-fl and CYP116B5-hd showing 10- and 3-folds higher activity, in terms of p-nitrocatechol produced per mg of enzyme per minute. CYP116B5-SOX represents an optimal model to exploit CYP116B5 and the same protein engineering approach could be used for P450s of the same class.

Enhanced and specific epoxidation activity of P450 BM3 mutants for the production of high value terpene derivatives.

Terpenes are natural molecules of valuable interest for different industrial applications. Cytochromes P450 enzymes can functionalize terpenoids to form high value oxidized derivatives in a green and sustainable manner, representing a valid alternative to chemical catalysis. In this work, an enhanced and specific epoxidation activity of cytochrome P450 BM3 mutants was found for the terpenes geraniol and linalool. This is the first report showing the epoxidation of linalool by P450 BM3 and its mutant A2 (Asp251Gly/Gln307His) with the formation of valuable oxide derivatives, highlighting the relevance of this enzymes for industrial applications.

Design of a H2 O2 -generating P450SPα fusion protein for high yield fatty acid conversion.

Sphingomonas paucimobilis' P450SPα (CYP152B1) is a good candidate as industrial biocatalyst. This enzyme is able to use hydrogen peroxide as unique cofactor to catalyze the fatty acids conversion to α-hydroxy fatty acids, thus avoiding the use of expensive electron-donor(s) and redox partner(s). Nevertheless, the toxicity of exogenous H2 O2 toward proteins and cells often results in the failure of the reaction scale-up when it is directly added as co-substrate. In order to bypass this problem, we designed a H2 O2 self-producing enzyme by fusing the P450SPα to the monomeric sarcosine oxidase (MSOX), as H2 O2 donor system, in a unique polypeptide chain, obtaining the P450SPα -polyG-MSOX fusion protein. The purified P450SPα -polyG-MSOX protein displayed high purity (A417 /A280  = 0.6) and H2 O2 -tolerance (kdecay  = 0.0021 ± 0.000055 min-1 ; ΔA417  = 0.018 ± 0.001) as well as good thermal stability (Tm : 59.3 ± 0.3°C and 63.2 ± 0.02°C for P450SPα and MSOX domains, respectively). The data show how the catalytic interplay between the two domains can be finely regulated by using 500 mM sarcosine as sacrificial substrate to generate H2 O2 . Indeed, the fusion protein resulted in a high conversion yield toward fat waste biomass-representative fatty acids, that is, lauric acid (TON = 6,800 compared to the isolated P450SPα TON = 2,307); myristic acid (TON = 6,750); and palmitic acid (TON = 1,962).

Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

Human aromatase is the cytochrome P450 catalyzing the conversion of androgens into estrogens in a three steps reaction essential to maintain steroid hormones balance. Here we report the capture and spectroscopic characterization of its compound I (Cpd I), the main reactive species in cytochromes P450. The typical spectroscopic transitions indicating the formation of Cpd I are detected within 0.8 s when mixing aromatase with meta-chloroperoxybenzoic acid. The estrogen product is obtained from the same reaction mixture, demonstrating the involvement of Cpd I in aromatization reaction. Site-directed mutagenesis is applied to the acid-alcohol pair D309 and T310 and to R192, predicted to be part of the proton relay network. Mutants D309N and R192Q do not lead to Cpd I with an associated loss of activity, confirming that these residues are involved in proton delivery for Cpd I generation. Cpd I is captured for T310A mutant and shows 2.9- and 4.4-fold faster rates of formation and decay, respectively, compared to wild-type (WT). However, its activity is lower than the WT and a larger amount of H2 O2 is produced during catalysis, indicating that T310 has an essential role in proton gating for generation of Cpd 0 and Cpd I and for their stabilization. The data provide new evidences on the role of threonine belonging to the conserved "acid-alcohol" pair and known to be crucial for oxygen activation in cytochromes P450.

Molecular Lego of Human Cytochrome P450: The Key Role of Heme Domain Flexibility for the Activity of the Chimeric Proteins.

The cytochrome P450 superfamily are heme-thiolate enzymes able to carry out monooxygenase reactions. Several studies have demonstrated the feasibility of using a soluble bacterial reductase from Bacillus megaterium, BMR, as an artificial electron transfer partner fused to the human P450 domain in a single polypeptide chain in an approach known as 'molecular Lego'. The 3A4-BMR chimera has been deeply characterized biochemically for its activity, coupling efficiency, and flexibility by many different biophysical techniques leading to the conclusion that an extension of five glycines in the loop that connects the two domains improves all the catalytic parameters due to improved flexibility of the system. In this work, we extend the characterization of 3A4-BMR chimeras using differential scanning calorimetry to evaluate stabilizing role of BMR. We apply the 'molecular Lego' approach also to CYP19A1 (aromatase) and the data show that the activity of the chimeras is very low (<0.003 min−1) for all the constructs tested with a different linker loop length: ARO-BMR, ARO-BMR-3GLY, and ARO-BMR-5GLY. Nevertheless, the fusion to BMR shows a remarkable effect on thermal stability studied by differential scanning calorimetry as indicated by the increase in Tonset by 10 °C and the presence of a cooperative unfolding process driven by the BMR protein domain. Previously characterized 3A4-BMR constructs show the same behavior of ARO-BMR constructs in terms of thermal stabilization but a higher activity as a function of the loop length. A comparison of the ARO-BMR system to 3A4-BMR indicates that the design of each P450-BMR chimera should be carefully evaluated not only in terms of electron transfer, but also for the biophysical constraints that cannot always be overcome by chimerization.

EPR characterization of the heme domain of a self-sufficient cytochrome P450 (CYP116B5).

CYP116B5 is a self-sufficient cytochrome P450 (CYP450) with interesting catalytic properties for synthetic purposes. When isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Here, by means of continuous wave and advanced EPR techniques, the coordination environment of iron in the isolated CYP116B5 heme domain (CYP116b5hd) is characterized. The ligand-free protein shows the characteristic EPR spectrum of a low-spin (S = 1/2) FeIII-heme with [gz = 2.440 ± 0.005, gy = 2.25 ± 0.01, gx = 1.92 ± 0.01]. These g-values reflect an electronic ground state very similar to classical P450 monooxygenases rather than P450 peroxygenases. Binding of imidazole results in g-values very close to the ones reported for CYP152 peroxygenases. The detection of hyperfine interactions through HYperfine Sub-level CORrElation (HYSCORE) Spectroscopy experiments, shows that this is due to a nitrogen-mediated axial coordination. This work adds a piece of experimental evidence to the research, aimed at elucidating the features that distinguish the classical P450 enzymes from peroxygenases. It shows that the electronic environment of heme iron of CYP116B5 in the resting state is similar to the classical P450 monooxygenases. Therefore, it is not the critical factor that confers to CYP116B5hd its peroxygenase-like activity, suggesting a crucial role of the protein matrix.

Assessment of Five Pesticides as Endocrine-Disrupting Chemicals: Effects on Estrogen Receptors and Aromatase.

Pesticides are widely applied all over the world, and pesticide exposure can induce different biological effects posing a possible threat to human health. Due to their effects on the endocrine system, some pesticides are classified as endocrine disruptors. The aim of the study is to assess the interference of five pesticides on estrogen biosynthesis and estrogen signaling. Three neonicotinoid insecticides (Acetamiprid, Clothianidin, and Thiamethoxam), a carbamate insecticide (Methiocarb) and a herbicide (Oxadiazon) were tested. The effect of pesticides on estrogen biosynthesis was studied through an ELISA assay using a recombinant form of human aromatase, the enzyme that catalyzes the transformation of androgens to estrogens. Moreover, the effect of pesticides on estrogen signaling was assessed using a gene reporter assay on MELN cells, which measures estrogen receptor-mediated estrogenic activity. The results of the ELISA assay showed that the pesticides did not alter aromatase activity (no interference with estrogen biosynthesis), while the results of the gene reporter assay showed that only Methiocarb was able to alter estrogen signaling at high doses. The estrogenic activity of Methiocarb, expressed as 17ß-estradiol equivalency factor (EEF), was equal to 8.0 × 10-8. In conclusion, this study suggested that Methiocarb should be considered a potential endocrine disruptor.

Heptad stereotypy, S/Q layering, and remote origin of the SARS-CoV-2 fusion core.

The fusion of the SARS-CoV-2 virus with cells, a key event in the pathogenesis of Covid-19, depends on the assembly of a six-helix fusion core (FC) formed by portions of the spike protein heptad repeats (HRs) 1 and 2. Despite the critical role in regulating infectivity, its distinctive features, origin, and evolution are scarcely understood. Thus, we undertook a structure-guided positional and compositional analysis of the SARS-CoV-2 FC, in comparison with FCs of related viruses, tracing its origin and ongoing evolution. We found that clustered amino acid substitutions within HR1, distinguishing SARS-CoV-2 from SARS-CoV-1, enhance local heptad stereotypy and increase sharply the FC serine-to-glutamine (S/Q) ratio, determining a neat alternate layering of S-rich and Q-rich subdomains along the post-fusion structure. Strikingly, SARS-CoV-2 ranks among viruses with the highest FC S/Q ratio, together with highly syncytiogenic respiratory pathogens (RSV, NDV), whereas MERS-Cov, HIV, and Ebola viruses display low ratios, and this feature reflects onto S/Q segregation and H-bonding patterns. Our evolutionary analyses revealed that the SARS-CoV-2 FC occurs in other SARS-CoV-1-like Sarbecoviruses identified since 2005 in Hong Kong and adjacent regions, tracing its origin to >50 years ago with a recombination-driven spread. Finally, current mutational trends show that the FC is varying especially in the FC1 evolutionary hotspot. These findings establish a novel analytical framework illuminating the sequence/structure evolution of the SARS-CoV-2 FC, tracing its long history within Sarbecoviruses, and may help rationalize the evolution of the fusion machinery in emerging pathogens and the design of novel therapeutic fusion inhibitors.

Synthesis of α-Hydroxy Fatty Acids from Fatty Acids by Intermediate α-Chlorination with TCCA under Solvent-Free Conditions: A Way to Valorization of Waste Fat Biomasses.

Within food wastes, including edible and inedible parts, fat biomasses represent a significant portion, often uneconomically used or improperly disposed causing pollution issues. Interesting perspectives for their management and valorization could be opened by conversion of fatty acids (FAs), which are their main constituents, into α-hydroxy FAs (α-HFAs), fine chemicals of great, but largely untapped potential, possibly due to current poor availability. Here, a simple and efficient procedure is reported to α-chlorinate FAs with trichloroisocyanuric acid (TCCA), a green halogenating agent, under solvent-free conditions and to directly convert the resultant α-chloro FAs, without previous purification, into α-HFAs. The procedure was applied to stearic, palmitic, and myristic acid and, with analogous success, to their mixture, ad hoc created to simulate a FAs mixture obtainable from a fat biomass.

Polymorphism on human aromatase affects protein dynamics and substrate binding: spectroscopic evidence.

Human aromatase is a member of the cytochrome P450 superfamily, involved in steroid hormones biosynthesis. In particular, it converts androgen into estrogens being therefore responsible for the correct sex steroids balance. Due to its capacity in producing estrogens it has also been considered as a promising target for breast cancer therapy. Two single-nucleotide polymorphisms (R264C and R264H) have been shown to alter aromatase activity and they have been associated to an increased or decreased risk for estrogen-dependent pathologies. Here, the effect of these mutations on the protein dynamics is investigated by UV/FTIR and time resolved fluorescence spectroscopy. H/D exchange rates were measured by FTIR for the three proteins in the ligand-free, substrate- and inhibitor-bound forms and the data indicate that the wild-type enzyme undergoes a conformational change leading to a more compact tertiary structure upon substrate or inhibitor binding. Indeed, the H/D exchange rates are decreased when a ligand is present. In the variants, the exchange rates in the ligand-free and -bound forms are similar, indicating that a structural change is lacking, despite the single amino acid substitution is located in the peripheral shell of the protein molecule. Moreover, the fluorescence lifetimes data show that the quenching effect on tryptophan-224 observed upon ligand binding in the wild-type, is absent in both variants. Since this residue is located in the catalytic pocket, these findings suggest that substrate entrance and/or retention in the active site is partially compromised in both mutants. A contact network analysis demonstrates that the protein structure is organized in two main clusters, whose connectivity is altered by ligand binding, especially in correspondence of helix-G, where the amino acid substitutions occur. Our findings demonstrate that SNPs resulting in mutations on aromatase surface modify the protein flexibility that is required for substrate binding and catalysis. The cluster analysis provides a rationale for such effect, suggesting helix G as a possible target for aromatase inhibition.

Self-Sufficient Class VII Cytochromes P450: From Full-Length Structure to Synthetic Biology Applications.

Members of class VII cytochromes P450 are catalytically self-sufficient enzymes containing a phthalate dioxygenase reductase-like domain fused to the P450 catalytic domain. Among these, CYP116B46 is the first enzyme for which the 3D structure of the whole polypeptide chain has been solved, shedding light on the interaction between its domains, which is crucial for catalysis. Most of these enzymes have been isolated from extremophiles or detoxifying bacteria that can carry out regio- and enantioselective oxidation of compounds of biotechnological interest. Protein engineering has generated mutants that can perform challenging organic reactions such as the anti-Markovnikov alkene oxidation. This potential, combined with the detailed 3D structure, forms the basis for further directed evolution studies aimed at widening their biotechnological exploitation.

Molecular and Structural Evolution of Cytochrome P450 Aromatase.

Aromatase is the cytochrome P450 enzyme converting androgens into estrogen in the last phase of steroidogenesis. As estrogens are crucial in reproductive biology, aromatase is found in vertebrates and the invertebrates of the genus Branchiostoma, where it carries out the aromatization reaction of the A-ring of androgens that produces estrogens. Here, we investigate the molecular evolution of this unique and highly substrate-selective enzyme by means of structural, sequence alignment, and homology modeling, shedding light on its key role in species conservation. The alignments led to the identification of a core structure that, together with key and unique amino acids located in the active site and the substrate recognition sites, has been well conserved during evolution. Structural analysis shows what their roles are and the reason why they have been preserved. Moreover, the residues involved in the interaction with the redox partner and some phosphorylation sites appeared late during evolution. These data reveal how highly substrate-selective cytochrome P450 has evolved, indicating that the driving forces for evolution have been the optimization of the interaction with the redox partner and the introduction of phosphorylation sites that give the possibility of modulating its activity in a rapid way.

Molecular Basis for Endocrine Disruption by Pesticides Targeting Aromatase and Estrogen Receptor.

The intensive use of pesticides has led to their increasing presence in water, soil, and agricultural products. Mounting evidence indicates that some pesticides may be endocrine disrupting chemicals (EDCs), being therefore harmful for the human health and the environment. In this study, three pesticides, glyphosate, thiacloprid, and imidacloprid, were tested for their ability to interfere with estrogen biosynthesis and/or signaling, to evaluate their potential action as EDCs. Among the tested compounds, only glyphosate inhibited aromatase activity (up to 30%) via a non-competitive inhibition or a mixed inhibition mechanism depending on the concentration applied. Then, the ability of the three pesticides to induce an estrogenic activity was tested in MELN cells. When compared to 17ß-estradiol, thiacloprid and imidacloprid induced an estrogenic activity at the highest concentrations tested with a relative potency of 5.4 × 10-10 and 3.7 × 10-9, respectively. Molecular dynamics and docking simulations predicted the potential binding sites and the binding mode of the three pesticides on the structure of the two key targets, providing a rational for their mechanism as EDCs. The results demonstrate that the three pesticides are potential EDCs as glyphosate acts as an aromatase inhibitor, whereas imidacloprid and thiacloprid can interfere with estrogen induced signaling.

Effector role of cytochrome P450 reductase for androstenedione binding to human aromatase.

Cytochromes P450 constitute a large superfamily of monooxygenases involved in many metabolic pathways. Most of them are not self-sufficient and need a reductase protein to provide the electrons necessary for catalysis. It was shown that the redox partner plays a role in the modulation of the structure and function of some bacterial P450 enzymes. Here, the effect of NADPH-cytochrome reductase (CPR) on human aromatase (Aro) is studied for what concerns its role in substrate binding. Pre-steady-state kinetic experiments indicate that both the substrate binding rates and the percentage of spin shift detected for aromatase are increased when CPR is present. Moreover, aromatase binds the substrate through a conformational selection mechanism, suggesting a possible effector role of CPR. The thermodynamic parameters for the formation of the CPR-Aro complex were studied by isothermal titration calorimetry. The dissociation constant of the complex formation is 4.5 folds lower for substrate-free compared to the substrate-bound enzyme. The enthalpy change observed when the CPR-Aro complex forms in the absence of the substrate are higher than in its presence, indicating that more interactions are formed/broken in the former case. Taken together, our data confirm that CPR has a role in promoting aromatase conformation optimal for substrate binding.

Chimeric cytochrome P450 3A4 used for in vitro prediction of food-drug interactions.

Inhibition of cytochrome P450 (CYP)-mediated drug metabolism by dietary substances is the main cause of drug-food interactions in humans. The present study reports on the in vitro inhibition assays of human CYP3A4 genetically linked to the reductase domain of bacterial BM3 of Bacillus megaterium (BMR) resulting in the chimeric protein CYP3A4-BMR. The activity of this chimeric enzyme was initially measured colorimetrically with erythromycin as the substrate where KM values similar to published data were determined. Subsequently, the inhibition assays with three different dietary products, grapefruit juice, curcumin, and resveratrol, were carried out with the chimeric enzyme both in solution and immobilized on electrode surfaces. For the solution studies, nicotinamide adenine dinucleotide phosphate was added as the electron donor, whereas the need for this cofactor was obviated in the immobilized enzyme as it was supplied by the electrode. Inhibition of the N-demethylation of erythromycin by CYP3A4-BMR chimera was measured at increasing concentrations of the different dietary compounds with calculated IC50 values of 0.5%, 31 µM, and 250 µM for grapefruit juice, curcumin, and resveratrol measured in solution compared with 0.7%, 24 µM, and 208 µM measured electrochemically, respectively. These data demonstrate the feasibility of the use of both CYP3A4-BMR chimera as well as bioelectrochemistry for in vitro studies of not only drug-food interactions but also prediction of adverse drug reactions in this important P450 enzyme.

Natural Compounds as Pharmaceuticals: The Key Role of Cytochromes P450 Reactivity.

The design of drugs from natural products is a re-emerging area due to the need for bioactive compounds. The exploitation of natural products and their derivatives obtained by biocatalysis is in line with the higher attention given today to new sustainable technologies that better preserve the environment (green chemistry). The research field of cytochromes P450 (CYPs) is continuously providing new enzymes and mutants that produce metabolites suitable for late-stage functionalization for new potential drugs. This review provides an overview of the exploitation of CYPs as biocatalysts in drug synthesis. Additionally, recent progress in protein and metabolic engineering is provided to show how these enzymes offer a toolbox that can be combined with other biocatalytic or chemical processes to build new platforms for the green production of new drugs.