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13 pages, 9986 KiB

Open AccessArticle

A Conserved Allosteric Site on Drug-Metabolizing CYPs: A Systematic Computational Assessment

by André Fischer and Martin Smieško

Int. J. Mol. Sci. 2021, 22(24), 13215; https://doi.org/10.3390/ijms222413215 - 8 Dec 2021

Cited by 11 | Viewed by 2692

Abstract

Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted [...] Read more.

Cytochrome P450 enzymes (CYPs) are the largest group of enzymes involved in human drug metabolism. Ligand tunnels connect their active site buried at the core of the membrane-anchored protein to the surrounding solvent environment. Recently, evidence of a superficial allosteric site, here denoted as hotspot 1 (H1), involved in the regulation of ligand access in a soluble prokaryotic CYP emerged. Here, we applied multi-scale computational modeling techniques to study the conservation and functionality of this allosteric site in the nine most relevant mammalian CYPs responsible for approximately 70% of drug metabolism. In total, we systematically analyzed over 44

μ

s of trajectories from conventional MD, cosolvent MD, and metadynamics simulations. Our bioinformatic analysis and simulations with organic probe molecules revealed the site to be well conserved in the CYP2 family with the exception of CYP2E1. In the presence of a ligand bound to the H1 site, we could observe an enlargement of a ligand tunnel in several members of the CYP2 family. Further, we could detect the facilitation of ligand translocation by H1 interactions with statistical significance in CYP2C8 and CYP2D6, even though all other enzymes except for CYP2C19, CYP2E1, and CYP3A4 presented a similar trend. As the detailed comprehension of ligand access and egress phenomena remains one of the most relevant challenges in the field, this work contributes to its elucidation and ultimately helps in estimating the selectivity of metabolic transformations using computational techniques. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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28 pages, 3715 KiB

Open AccessArticle

Developmental Toxicity and Biotransformation of Two Anti-Epileptics in Zebrafish Embryos and Early Larvae

by Chloé Bars, Jente Hoyberghs, Allan Valenzuela, Laura Buyssens, Miriam Ayuso, Chris Van Ginneken, Alain J. Labro, Kenn Foubert and Steven J. Van Cruchten

Int. J. Mol. Sci. 2021, 22(23), 12696; https://doi.org/10.3390/ijms222312696 - 24 Nov 2021

Cited by 9 | Viewed by 3993

Abstract

The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and [...] Read more.

The zebrafish (Danio rerio) embryo is gaining interest as a bridging tool between in-vitro and in-vivo developmental toxicity studies. However, cytochrome P450 (CYP)-mediated drug metabolism in this model is still under debate. Therefore, we investigated the potential of zebrafish embryos and larvae to bioactivate two known anti-epileptics, carbamazepine (CBZ) and phenytoin (PHE), to carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH), respectively. First, zebrafish were exposed to CBZ, PHE, E-CBZ and HPPH from 5¼- to 120-h post fertilization (hpf) and morphologically evaluated. Second, the formations of E-CBZ and HPPH were assessed in culture medium and in whole-embryo extracts at different time points by targeted LC-MS. Finally, E-CBZ and HPPH formation was also assessed in adult zebrafish liver microsomes and compared with those of human, rat, and rabbit. The present study showed teratogenic effects for CBZ and PHE, but not for E-CBZ and HPPH. No HPPH was detected during organogenesis and E-CBZ was only formed at the end of organogenesis. E-CBZ and HPPH formation was also very low-to-negligible in adult zebrafish compared with the mammalian species. As such, other metabolic pathways than those of mammals are involved in the bioactivation of CBZ and PHE, or, these anti-epileptics are teratogens and do not require bioactivation in the zebrafish. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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23 pages, 7316 KiB

Open AccessArticle

Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta

by Aiti Vizzini, Angela Bonura, Laura La Paglia, Antonino Fiannaca, Massimo La Rosa, Alfonso Urso, Manuela Mauro, Mirella Vazzana and Vincenzo Arizza

Int. J. Mol. Sci. 2021, 22(20), 11141; https://doi.org/10.3390/ijms222011141 - 15 Oct 2021

Cited by 7 | Viewed by 1814

Abstract

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In [...] Read more.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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9 pages, 3614 KiB

Open AccessCommunication

The FMN “140s Loop” of Cytochrome P450 Reductase Controls Electron Transfer to Cytochrome P450

by Freeborn Rwere, Sangchoul Im and Lucy Waskell

Int. J. Mol. Sci. 2021, 22(19), 10625; https://doi.org/10.3390/ijms221910625 - 30 Sep 2021

Cited by 2 | Viewed by 2074

Abstract

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. [...] Read more.

Cytochrome P450 reductase (CYPOR) provides electrons to all human microsomal cytochrome P450s (cyt P450s). The length and sequence of the “140s” FMN binding loop of CYPOR has been shown to be a key determinant of its redox potential and activity with cyt P450s. Shortening the “140s loop” by deleting glycine-141(ΔGly141) and by engineering a second mutant that mimics flavo-cytochrome P450 BM3 (ΔGly141/Glu142Asn) resulted in mutants that formed an unstable anionic semiquinone. In an attempt to understand the molecular basis of the inability of these mutants to support activity with cyt P450, we expressed, purified, and determined their ability to reduce ferric P450. Our results showed that the ΔGly141 mutant with a very mobile loop only reduced ~7% of cyt P450 with a rate similar to that of the wild type. On the other hand, the more stable loop in the ΔGly141/Glu142Asn mutant allowed for ~55% of the cyt P450 to be reduced ~60% faster than the wild type. Our results reveal that the poor activity of the ΔGly141 mutant is primarily accounted for by its markedly diminished ability to reduce ferric cyt P450. In contrast, the poor activity of the ΔGly141/Glu142Asn mutant is presumably a consequence of the altered structure and mobility of the “140s loop”. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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22 pages, 8861 KiB

Open AccessArticle

Deciphering Structural Alterations Associated with Activity Reductions of Genetic Polymorphisms in Cytochrome P450 2A6 Using Molecular Dynamics Simulations

by Koichi Kato, Tomoki Nakayoshi, Rika Nokura, Hiroki Hosono, Masahiro Hiratsuka, Yoshinobu Ishikawa, Eiji Kurimoto and Akifumi Oda

Int. J. Mol. Sci. 2021, 22(18), 10119; https://doi.org/10.3390/ijms221810119 - 19 Sep 2021

Cited by 3 | Viewed by 2548

Abstract

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities [...] Read more.

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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16 pages, 1936 KiB

Open AccessArticle

In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

by Olufunmilayo Olukemi Akapo, Joanna M. Macnar, Justyna D. Kryś, Puleng Rosinah Syed, Khajamohiddin Syed and Dominik Gront

Int. J. Mol. Sci. 2021, 22(15), 7811; https://doi.org/10.3390/ijms22157811 - 22 Jul 2021

Cited by 5 | Viewed by 2502

Abstract

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance [...] Read more.

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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19 pages, 21829 KiB

Open AccessArticle

Single Nucleotide Polymorphism Induces Divergent Dynamic Patterns in CYP3A5: A Microsecond Scale Biomolecular Simulation of Variants Identified in Sub-Saharan African Populations

by Houcemeddine Othman, Jorge E. B. da Rocha and Scott Hazelhurst

Int. J. Mol. Sci. 2021, 22(15), 7786; https://doi.org/10.3390/ijms22157786 - 21 Jul 2021

Cited by 2 | Viewed by 2454

Abstract

Pharmacogenomics aims to reveal variants associated with drug response phenotypes. Genes whose roles involve the absorption, distribution, metabolism, and excretion of drugs, are highly polymorphic between populations. High coverage whole genome sequencing showed that a large proportion of the variants for these genes [...] Read more.

Pharmacogenomics aims to reveal variants associated with drug response phenotypes. Genes whose roles involve the absorption, distribution, metabolism, and excretion of drugs, are highly polymorphic between populations. High coverage whole genome sequencing showed that a large proportion of the variants for these genes are rare in African populations. This study investigated the impact of such variants on protein structure to assess their functional importance. We used genetic data of CYP3A5 from 458 individuals from sub-Saharan Africa to conduct a structural bioinformatics analysis. Five missense variants were modeled and microsecond scale molecular dynamics simulations were conducted for each, as well as for the CYP3A5 wildtype and the Y53C variant, which has a known deleterious impact on enzyme activity. The binding of ritonavir and artemether to CYP3A5 variant structures was also evaluated. Our results showed different conformational characteristics between all the variants. No significant structural changes were noticed. However, the genetic variability seemed to act on the plasticity of the protein. The impact on drug binding might be drug dependant. We concluded that rare variants hold relevance in determining the pharmacogenomics properties of populations. This could have a significant impact on precision medicine applications in sub-Saharan Africa. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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16 pages, 5746 KiB

Open AccessArticle

Structural Basis for the Diminished Ligand Binding and Catalytic Ability of Human Fetal-Specific CYP3A7

by Irina F. Sevrioukova

Int. J. Mol. Sci. 2021, 22(11), 5831; https://doi.org/10.3390/ijms22115831 - 29 May 2021

Cited by 8 | Viewed by 2552

Abstract

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are [...] Read more.

Cytochrome P450 3A7 (CYP3A7) is a fetal/neonatal liver enzyme that participates in estriol synthesis, clearance of all-trans retinoic acid, and xenobiotic metabolism. Compared to the closely related major drug-metabolizing enzyme in adult liver, CYP3A4, the ligand binding and catalytic capacity of CYP3A7 are substantially reduced. To better understand the structural basis for these functional differences, the 2.15 Å crystal structure of CYP3A7 has been solved. Comparative analysis of CYP3A enzymes shows that decreased structural plasticity rather than the active site microenvironment defines the ligand binding ability of CYP3A7. In particular, a rotameric switch in the gatekeeping amino acid F304 triggers local and long-range rearrangements that transmit to the F-G fragment and alter its interactions with the I-E-D-helical core, resulting in a more rigid structure. Elongation of the β₃-β₄ strands, H-bond linkage in the substrate channel, and steric constraints in the C-terminal loop further increase the active site rigidity and limit conformational ensemble. Collectively, these structural distinctions lower protein plasticity and change the heme environment, which, in turn, could impede the spin-state transition essential for optimal reactivity and oxidation of substrates. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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14 pages, 21150 KiB

Open AccessArticle

Conformational Landscape of Cytochrome P450 Reductase Interactions

by Manuel Sellner, André Fischer, Charleen G. Don and Martin Smieško

Int. J. Mol. Sci. 2021, 22(3), 1023; https://doi.org/10.3390/ijms22031023 - 20 Jan 2021

Cited by 13 | Viewed by 3793

Abstract

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a [...] Read more.

Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein–protein docking to investigate the conformational changes necessary for the formation of the CPR–CYP complex. While some structural features of the CPR and the CPR–CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR–CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR–CYP interactions and underlying conformational changes, improving our understanding of this complex machinery relevant for drug metabolism. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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22 pages, 5313 KiB

Open AccessArticle

Rational Design of CYP3A4 Inhibitors: A One-Atom Linker Elongation in Ritonavir-Like Compounds Leads to a Marked Improvement in the Binding Strength

by Eric R. Samuels and Irina F. Sevrioukova

Int. J. Mol. Sci. 2021, 22(2), 852; https://doi.org/10.3390/ijms22020852 - 16 Jan 2021

Cited by 14 | Viewed by 3185

Abstract

Inhibition of the major human drug-metabolizing cytochrome P450 3A4 (CYP3A4) by pharmaceuticals and other xenobiotics could lead to toxicity, drug–drug interactions and other adverse effects, as well as pharmacoenhancement. Despite serious clinical implications, the structural basis and attributes required for the potent inhibition [...] Read more.

Inhibition of the major human drug-metabolizing cytochrome P450 3A4 (CYP3A4) by pharmaceuticals and other xenobiotics could lead to toxicity, drug–drug interactions and other adverse effects, as well as pharmacoenhancement. Despite serious clinical implications, the structural basis and attributes required for the potent inhibition of CYP3A4 remain to be established. We utilized a rational inhibitor design to investigate the structure–activity relationships in the analogues of ritonavir, the most potent CYP3A4 inhibitor in clinical use. This study elucidated the optimal length of the head-group spacer using eleven (series V) analogues with the R₁/R₂ side-groups as phenyls or R₁–phenyl/R₂–indole/naphthalene in various stereo configurations. Spectral, functional and structural characterization of the inhibitory complexes showed that a one-atom head-group linker elongation, from pyridyl–ethyl to pyridyl–propyl, was beneficial and markedly improved K_s, IC₅₀ and thermostability of CYP3A4. In contrast, a two-atom linker extension led to a multi-fold decrease in the binding and inhibitory strength, possibly due to spatial and/or conformational constraints. The lead compound, 3h, was among the best inhibitors designed so far and overall, the strongest binder (K_s and IC₅₀ of 0.007 and 0.090 µM, respectively). 3h was the fourth structurally simpler inhibitor superior to ritonavir, which further demonstrates the power of our approach. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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12 pages, 2805 KiB

Open AccessArticle

Dose-Dependent Effects of Resveratrol on Cisplatin-Induced Hearing Loss

by Chang Ho Lee, Kyung Woon Kim, So Min Lee and So Young Kim

Int. J. Mol. Sci. 2021, 22(1), 113; https://doi.org/10.3390/ijms22010113 - 24 Dec 2020

Cited by 16 | Viewed by 2724

Abstract

Previous preclinical studies have demonstrated the otoprotective effects of resveratrol (RV) at low doses. This study aimed to investigate the dose-dependent effects of RV in rats with cisplatin (CXP)-induced hearing loss. Sprague-Dawley rats (8-weeks old) were divided into six treatment groups (n [...] Read more.

Previous preclinical studies have demonstrated the otoprotective effects of resveratrol (RV) at low doses. This study aimed to investigate the dose-dependent effects of RV in rats with cisplatin (CXP)-induced hearing loss. Sprague-Dawley rats (8-weeks old) were divided into six treatment groups (n = 12/group) and treated as follows: control, 0.5 mg/kg RV, 50 mg/kg RV, CXP, 0.5 mg/kg RV + CXP), and 50 mg/kg RV + CXP groups. CXP (3 mg/kg) was intraperitoneally injected for 5 days. RV (0.5 or 50 mg/kg) was intraperitoneally injected for 10 days from the first day of CXP administration. Auditory brainstem response (ABR) thresholds were measured before and within 3 days at the end of the drug administration. Cochlear tissues were harvested, and the outer hair cells were examined using cochlear whole mounts. The mRNA expression of NFκB, IL6, IL1β, and CYP1A1, and protein levels of aryl hydrocarbon receptor (AhR) and cytosolic and nuclear receptor for advanced glycation endproducts (RAGE) were evaluated. The ABR threshold increased in the 50 mg/kg RV and CXP groups at 4, 8, 16, and 32 kHz. The 0.5 mg/kg RV + CXP group demonstrated decreased hearing thresholds at 4 and 32 kHz compared to the CXP group. Cochlear whole-mount analysis revealed loss of outer hair cells in the 50 mg/kg RV and CXP groups and partial prevention of these cells in the 0.5 mg/kg RV + CXP group. The mRNA expressions of NFκB, IL6, and IL1β were increased in the 50 mg/kg RV and CXP groups compared to the control group. In contrast, these levels were decreased in the 0.5 mg/kg RV + CXP group compared to the CXP group. The mRNA expression of CYP1A1 was increased in the CXP group, while it was decreased in the 0.5 mg/kg RV + CXP group compared to the control group. The protein levels of AhR and cytosolic RAGE decreased in the 0.5 mg/kg RV group. Low-dose RV had partial otoprotective effects on CXP ototoxicity. The otoprotective effects of RV may be mediated through anti-oxidative (CYP1A1 and RAGE) and anti-inflammatory (NFκB, IL6, and IL1β) responses. High-dose RV exerted an inflammatory response and did not ameliorate CXP-induced ototoxicity. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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19 pages, 3425 KiB

Open AccessArticle

Interaction Modes of Microsomal Cytochrome P450s with Its Reductase and the Role of Substrate Binding

by Francisco Esteves, Philippe Urban, José Rueff, Gilles Truan and Michel Kranendonk

Int. J. Mol. Sci. 2020, 21(18), 6669; https://doi.org/10.3390/ijms21186669 - 11 Sep 2020

Cited by 12 | Viewed by 2414

Abstract

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is [...] Read more.

The activity of microsomal cytochromes P450 (CYP) is strictly dependent on the supply of electrons provided by NADPH cytochrome P450 oxidoreductase (CPR). The variant nature of the isoform-specific proximal interface of microsomal CYPs implies that the interacting interface between the two proteins is degenerated. Recently, we demonstrated that specific CPR mutations in the FMN-domain (FD) may induce a gain in activity for a specific CYP isoform. In the current report, we confirm the CYP isoform dependence of CPR’s degenerated binding by demonstrating that the effect of four of the formerly studied FD mutants are indeed exclusive of a specific CYP isoform, as verified by cytochrome c inhibition studies. Moreover, the nature of CYP’s substrate seems to have a modulating role in the CPR:CYP interaction. In silico molecular dynamics simulations of the FD evidence that mutations induces very subtle structural alterations, influencing the characteristics of residues formerly implicated in the CPR:CYP interaction or in positioning of the FMN moiety. CPR seems therefore to be able to form effective interaction complexes with its structural diverse partners via a combination of specific structural features of the FD, which are functional in a CYP isoform dependent manner, and dependent on the substrate bound. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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Review

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32 pages, 4519 KiB

Open AccessReview

Monooxygenase- and Dioxygenase-Catalyzed Oxidative Dearomatization of Thiophenes by Sulfoxidation, cis-Dihydroxylation and Epoxidation

by Derek R. Boyd, Narain D. Sharma, Paul J. Stevenson, Patrick Hoering, Christopher C. R. Allen and Patrick M. Dansette

Int. J. Mol. Sci. 2022, 23(2), 909; https://doi.org/10.3390/ijms23020909 - 14 Jan 2022

Cited by 5 | Viewed by 2999

Abstract

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, [...] Read more.

Enzymatic oxidations of thiophenes, including thiophene-containing drugs, are important for biodesulfurization of crude oil and drug metabolism of mono- and poly-cyclic thiophenes. Thiophene oxidative dearomatization pathways involve reactive metabolites, whose detection is important in the pharmaceutical industry, and are catalyzed by monooxygenase (sulfoxidation, epoxidation) and dioxygenase (sulfoxidation, dihydroxylation) enzymes. Sulfoxide and epoxide metabolites of thiophene substrates are often unstable, and, while cis-dihydrodiol metabolites are more stable, significant challenges are presented by both types of metabolite. Prediction of the structure, relative and absolute configuration, and enantiopurity of chiral metabolites obtained from thiophene enzymatic oxidation depends on the substrate, type of oxygenase selected, and molecular docking results. The racemization and dimerization of sulfoxides, cis/trans epimerization of dihydrodiol metabolites, and aromatization of epoxides are all factors associated with the mono- and di-oxygenase-catalyzed metabolism of thiophenes and thiophene-containing drugs and their applications in chemoenzymatic synthesis and medicine. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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23 pages, 4510 KiB

Open AccessReview

A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

by Sian Thistlethwaite, Laura N. Jeffreys, Hazel M. Girvan, Kirsty J. McLean and Andrew W. Munro

Int. J. Mol. Sci. 2021, 22(21), 11380; https://doi.org/10.3390/ijms222111380 - 21 Oct 2021

Cited by 15 | Viewed by 4408

Abstract

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in [...] Read more.

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production. Full article

(This article belongs to the Special Issue Cytochromes P450: Drug Metabolism, Bioactivation and Biodiversity 3.0)

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