Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Single Mutations in Cytochrome P450 Oxidoreductase Can Alter the Specificity of Human Cytochrome P450 1A2-Mediated Caffeine Metabolism.

A unique cytochrome P450 (CYP) oxidoreductase (CPR) sustains activities of human microsomal CYPs. Its function requires toggling between a closed conformation enabling electron transfers from NADPH to FAD and then FMN cofactors and open conformations forming complexes and transferring electrons to CYPs. We previously demonstrated that distinct features of the hinge region linking the FAD and FMN domain (FD) modulate conformer poses and their interactions with CYPs. Specific FD residues contribute in a CYP isoform-dependent manner to the recognition and electron transfer mechanisms that are additionally modulated by the structure of CYP-bound substrate. To obtain insights into the underlying mechanisms, we analyzed how hinge region and FD mutations influence CYP1A2-mediated caffeine metabolism. Activities, metabolite profiles, regiospecificity and coupling efficiencies were evaluated in regard to the structural features and molecular dynamics of complexes bearing alternate substrate poses at the CYP active site. Studies reveal that FD variants not only modulate CYP activities but surprisingly the regiospecificity of reactions. Computational approaches evidenced that the considered mutations are generally in close contact with residues at the FD-CYP interface, exhibiting induced fits during complexation and modified dynamics depending on caffeine presence and orientation. It was concluded that dynamic coupling between FD mutations, the complex interface and CYP active site exist consistently with the observed regiospecific alterations.

Confrontation of AlphaFold models with experimental structures enlightens conformational dynamics supporting CYP102A1 functions.

Conformational dynamics plays a critical role for the function of multidomain electron transfer complexes. While crystallographic or NMR approaches allow detailed insight into structures, lower resolution methods like cryo-electron microscopy can provide more information on dynamics. In silico structure modelling using AlphaFold was recently successfully extended to the prediction of protein complexes but its capability to address large conformational changes involved in catalysis remained obscure. We used bacterial CYP102A1 monooxygenase homodimer as a test case to design a competitive modelling approach (CMA) for assessing alternate conformations of multi-domain complexes. Predictions were confronted with published crystallographic and cryo-EM data, evidencing consistencies but also permitting some reinterpretation of experimental data. Structural determinants stabilising the new type of domain connectivity evidenced in this bacterial self-sufficient monooxygenase were analysed by CMA and used for in silico retro-engineering applied to its eukaryotic bi-component counterparts.

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

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.

Corrigendum: The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

[This corrects the article DOI: 10.3389/fphar.2020.00299.].

The Role of the FMN-Domain of Human Cytochrome P450 Oxidoreductase in Its Promiscuous Interactions With Structurally Diverse Redox Partners.

NADPH cytochrome P450 oxidoreductase (CPR) is the obligatory electron supplier that sustains the activity of microsomal cytochrome P450 (CYP) enzymes. The variant nature of the isoform-specific proximal interface of microsomal CYPs indicates that CPR is capable of multiple degenerated interactions with CYPs for electron transfer, through different binding mechanisms, and which are still not well-understood. Recently, we showed that CPR dynamics allows formation of open conformations that can be sampled by its structurally diverse redox partners in a CYP-isoform dependent manner. To further investigate the role of the CPR FMN-domain in effective binding of CPR to its diverse acceptors and to clarify the underlying molecular mechanisms, five different CPR-FMN-domain random mutant libraries were created. These libraries were screened for mutants with increased activity when combined with specific CYP-isoforms. Seven CPR-FMN-domain mutants were identified, supporting a gain in activity for CYP1A2 (P117H, G144C, A229T), 2A6 (P117L/L125V, G175D, H183Y), or 3A4 (N151D). Effects were evaluated using extended enzyme kinetic analysis, cytochrome b 5 competition, ionic strength effect on CYP activity, and structural analysis. Mutated residues were located either in or adjacent to several acidic amino acid stretches - formerly indicated to be involved in CPR:CYP interactions - or close to two tyrosine residues suggested to be involved in FMN binding. Several of the identified positions co-localize with mutations found in naturally occurring CPR variants that were previously shown to cause CYP-isoform-dependent effects. The mutations do not seem to significantly alter the geometry of the FMN-domain but are likely to cause very subtle alterations leading to improved interaction with a specific CYP. Overall, these data suggest that CYPs interact with CPR using an isoform specific combination of several binding motifs of the FMN-domain.

Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae.

4-hydroxybenzoic acid (pHBA) is an important industrial precursor of muconic acid and liquid crystal polymers whose production is based on the petrochemical industry. In order to decrease our dependency on fossil fuels and improve sustainability, microbial engineering is a particularly appealing approach for replacing traditional chemical techniques. The optimization of microbial strains, however, is still highly constrained by the screening stage. Biosensors have helped to alleviate this problem by decreasing the screening time as well as enabling higher throughput. In this paper, we constructed a synthetic biosensor, named sBAD, consisting of a fusion of the pHBA-binding domain of HbaR from R. palustris, the LexA DNA binding domain at the N-terminus and the transactivation domain B112 at the C-terminus. The response of sBAD was tested in the presence of different benzoic acid derivatives, with cell fluorescence output measured by flow cytometry. The biosensor was found to be activated by the external addition of pHBA in the culture medium, in addition to other carboxylic acids including p-aminobenzoic acid (pABA), salicylic acid, anthranilic acid, aspirin, and benzoic acid. Furthermore, we were able to show that this biosensor could detect the in vivo production of pHBA in a genetically modified yeast strain. A good linearity was observed between the biosensor fluorescence and pHBA concentration. Thus, this biosensor would be well-suited as a high throughput screening tool to produce, via metabolic engineering, benzoic acid derivatives.

Probing the Role of the Hinge Segment of Cytochrome P450 Oxidoreductase in the Interaction with Cytochrome P450.

NADPH-cytochrome P450 reductase (CPR) is the unique redox partner of microsomal cytochrome P450s (CYPs). CPR exists in a conformational equilibrium between open and closed conformations throughout its electron transfer (ET) function. Previously, we have shown that electrostatic and flexibility properties of the hinge segment of CPR are critical for ET. Three mutants of human CPR were studied (S243P, I245P and R246A) and combined with representative human drug-metabolizing CYPs (isoforms 1A2, 2A6 and 3A4). To probe the effect of these hinge mutations different experimental approaches were employed: CYP bioactivation capacity of pre-carcinogens, enzyme kinetic analysis, and effect of the ionic strength and cytochrome b5 (CYB5) on CYP activity. The hinge mutations influenced the bioactivation of pre-carcinogens, which seemed CYP isoform and substrate dependent. The deviations of Michaelis-Menten kinetic parameters uncovered tend to confirm this discrepancy, which was confirmed by CYP and hinge mutant specific salt/activity profiles. CPR/CYB5 competition experiments indicated a less important role of affinity in CPR/CYP interaction. Overall, our data suggest that the highly flexible hinge of CPR is responsible for the existence of a conformational aggregate of different open CPR conformers enabling ET-interaction with structural varied redox partners.

Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4.

Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We report here on a bacterial whole-cells high-throughput method for the activity evaluation of human CYP1A2, 2A6, and 3A4, when sustained by NADPH cytochrome P450 oxidoreductase (CPR), in the absence or presence of cytochrome b5 (CYB5). This new assay consists of a microplate real-time fluorometric method, with direct measurement of metabolite formation, in a suspension of Escherichia coli BTC-CYP bacteria, a human CYP competent tester strain when incubated with specific fluorogenic substrates. Overall, the maximum turnover (kcat) velocities of the three human CYPs resulting from the whole-BTC cells assays were similar to those obtained when applying the corresponding standard reference membrane fractions assays. CYP activity screening with co-expression of CYB5 suggests an enhancing effect of CYB5 on the kcat of specific isoforms, when using the whole-BTC cells assay. Our results demonstrate that this new approach can offer an efficient high-throughput method for screening of CYP1A2, 2A6 and 3A4 activity and can be potentially applicable for other human CYPs. This can be of particular use for timely and efficient screening of chemical libraries or mutant libraries of CYP enzyme complex proteins, without the necessity for labor intensive isolation of subcellular fractions.

Ligand Access Channels in Cytochrome P450 Enzymes: A Review.

Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents different in silico tools that were developed to uncover such channels in P450 crystal structures. It also lists some of the experimental evidence that actually suggest that these predicted channels might indeed play a critical role in modulating P450 functions. Amino acid residues at the entrance of the channels may participate to a first global ligand recognition of ligands by P450 enzymes before they reach the buried active site. Moreover, different P450 enzymes show different networks of predicted channels. The plasticity of P450 structures is also important to take into account when looking at how channels might play their role.

Correction: The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.

[This corrects the article on p. 755 in vol. 8, PMID: 29163152.].

The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength.

NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners.

Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants.

Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level.

Ordered chimerogenesis applied to CYP2B P450 enzymes.

BACKGROUND: Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11. METHODS: A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast. RESULTS: Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels. CONCLUSIONS: Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity. GENERAL SIGNIFICANCE: The result provides a new basis for determining the structural elements that control functions in complex enzymes.

Functional characterization of zebrafish cytochrome P450 1 family proteins expressed in yeast.

BACKGROUND: Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast. METHODS: CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone. RESULTS: CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6ß-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles. CONCLUSIONS: Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds. GENERAL SIGNIFICANCE: Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.

Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes.

BACKGROUND: A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. METHODS: Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. RESULTS: The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. CONCLUSIONS: Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. GENERAL SIGNIFICANCE: Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site.

High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation.

Altered substrate specificity of the Pterygoplichthys sp. (Loricariidae) CYP1A enzyme.

Ethoxyresorufin is a classical substrate for vertebrate CYP1A enzymes. In Pterygoplichthys sp. (Loricariidae) this enzyme possesses 48 amino acids substitutions compared to CYP1A sequences from other vertebrate species. These substitutions or a certain subset substitution are responsible for the non-detection of the EROD reaction in this species liver microsomes. In the present study, we investigated the catalytic activity of Pterygoplichthys sp. CYP1A toward 15 potential substrates in order to understand the substrate preferences of this modified CYP1A. The fish gene was expressed in yeast and the accumulation of the protein was confirmed by both the characteristic P450-CO absorbance spectra and by detection with monoclonal antibodies. Catalytic activities were assayed with yeast microsomes and four resorufin ethers, six coumarin derivates, three flavones, resveratrol and ethoxyfluoresceinethylester. Results demonstrated that the initial velocity pattern of this enzyme for the resorufin derivatives is different from the one described for most vertebrate CYP1As. The initial velocity for the activity with the coumarin derivatives is several orders of magnitude higher than with the resorufins, i.e. the turnover number (kcat) for ECOD is 400× higher than for EROD. Nonetheless, the specificity constant (kcat/km) for EROD is only slightly higher than for ECOD. EFEE is degraded at a rate comparable to the resorufins. Pterygoplichthys sp. CYP1A also degrades 7-methoxyflavone and ß-naphthoflavone but not resveratrol and chrysin. These results indicate a divergent substrate preference for Pterygoplichthys sp. CYP1A, which may be involved in the adaptation of Loricariidae fish to their particular environment and feeding habits.

High-throughput enzymology and combinatorial mutagenesis for mining cytochrome P450 functions.

BACKGROUND: High-throughput (HT) characterization of drugs for potential biotransformation and interaction is routine in pharmaceutical industry. OBJECTIVE: HT approaches were extended to enzyme studies for identifying combinations of structural elements that control substrate specificity. METHODS: Structure-based and combinatorial mutagenesis have been applied with success to decipher P450 structure-function relationships. The idea is to measure activities on a library of combinatorial variants of similar structure with a large collection of substrates presenting a similar chemical scaffold. This combinatorial approach is then associated to multivariate statistics to relate functional features to structural determinants. RESULTS/CONCLUSION: A method to measure HT kinetics is presented. The proposed statistical approach is illustrated with tri- and tetracyclic substrates and artificial variant enzymes of the CYP1A subfamily.

A combinatorial approach to substrate discrimination in the P450 CYP1A subfamily.

A comparison of all known mammalian CYP1A sequences identifies nineteen sequence regions that are conserved within all 1A1s or within all 1A2s but at the same time systematically differ between any 1A1 and any 1A2. The purpose of this study was to explore links between these specific CYP1A sequence signatures and substrate specificity shift through the kinetic analysis of combinatorial variants of increasing complexity. The less complex variants correspond to multiple mutations within a short segment of their sequence. The more complex variants correspond to mosaic P450s recombining 1A1 and 1A2 sequences (up to 5 crossovers per sequence). Fifty-eight such functional CYP1A variants and parental wild-type enzymes were expressed in yeast and assayed with 7-alkoxyresorufins and ethoxyflurorescein ethyl ester as substrates. Observed kinetic data were analyzed by multivariate statistical analyses and hierarchical clustering in order to highlight correlations and identify potential sequence-activity relationships within the three-dimensional function space investigated. Several variants are outliers in these representations and show a redistribution of their substrate specificity compared to wild-type CYP1As. Some combinations of sequence elements were identified that significantly discriminate between 1A1 and 1A2 for these three substrates. The comparison of this combinatorial approach with previous results of site-directed mutagenesis is discussed.