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.

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.

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.

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.

A well-balanced preexisting equilibrium governs electron flux efficiency of a multidomain diflavin reductase.

Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains.

BACKGROUND: A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs - complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes. METHODS: An original algorithmic approach (Global Sequence Signature-GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences. RESULTS: Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation. CONCLUSIONS: Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. GENERAL SIGNIFICANCE: In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.

Characterization of chitinases from the GH18 gene family in the myxomycete Physarum polycephalum.

BACKGROUND: Physarum polycephalum is an unusual macroscopic myxomycete expressing a large range of glycosyl hydrolases. Among them, enzymes from the GH18 family can hydrolyze chitin, an important structural component of the cell walls in fungi and in the exoskeleton of insects and crustaceans. METHODS: Low stringency sequence signature search in transcriptomes was used to identify GH18 sequences related to chitinases. Identified sequences were expressed in E. coli and corresponding structures modelled. Synthetic substrates and in some cases colloidal chitin were used to characterize activities. RESULTS: Catalytically functional hits were sorted and their predicted structures compared. All share the TIM barrel structure of the GH18 chitinase catalytic domain, optionally fused to binding motifs, such as CBM50, CBM18, and CBM14, involved in sugar recognition. Assessment of the enzymatic activities following deletion of the C-terminal CBM14 domain of the most active clone evidenced a significant contribution of this extension to the chitinase activity. A classification based on module organization, functional and structural criteria of characterized enzymes was proposed. CONCLUSIONS: Physarum polycephalum sequences encompassing a chitinase like GH18 signature share a modular structure involving a structurally conserved catalytic TIM barrels decorated or not by a chitin insertion domain and optionally surrounded by additional sugar binding domains. One of them plays a clear role in enhancing activities toward natural chitin. GENERAL SIGNIFICANCE: Myxomycete enzymes are currently poorly characterized and constitute a potential source for new catalysts. Among them glycosyl hydrolases have a strong potential for valorization of industrial waste as well as in therapeutic field.

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.

Design and experimental validation of a generic model for combinatorial assembly of DNA tiles into 1D-structures.

BACKGROUND: Quantitative modeling of the self-assembly of DNA tiles leading either to defined end-products or distribution of biopolymers is of practical importance for biotechnology and synthetic biology. METHODS: The combinatorial process describing tile assembly was implemented into a generic algorithm allowing quantitative description of the population of significant species accumulating during the reaction course. Experimental formation and characterization by optical and electrophoresis approaches of copolymers resulting from the self-assembly of a limited number of half-complementary tiles were used to define and validate generic rules allowing definition of model parameters. RESULTS: Factors controlling the structure and the dynamic of the oligomer population were evidenced for assemblies leading or not to defined end-products. Primary parameters were experimentally determined using rapid mixing experiments. Adjustment of simulations to experimental profiles allowed definition of generic rules for calculation of secondary parameters that take into account macro- and microenvironment of individual hybridization steps. In the case of copolymers, accurate simulation of experimental profiles was achieved for formation of linear assemblies. CONCLUSIONS: Overall length of species and structure of the DNA regions flanking the hybridization sites are critical parameters for which calculation rules were defined. The computational approach quantitatively predicted the parameters affecting time-course and distribution of accumulating products for different experimental designs. GENERAL SIGNIFICANCE: The computational and parameter evaluation procedures designed for the assembly of DNA tiles into large 1D-structures are more generally applicable for the construction of non-DNA polymers by extremities-specific recognition of molecular blocks.

Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH-cytochrome P450 reductase.

CPR (NADPH-cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.

[C7GC4]4 association into supra molecular i-motif structures.

The self-associative properties of cytidine-rich oligonucleotides into symmetrical i-motif tetramers give to these oligonucleotides the capacity of forming supramolecular structures (sms) that have potential applications in the nanotechnology domain. In order to facilitate sms formation, oligonucleotides containing two cytidine stretches of unequal length (C(n)XC(m)) separated by a non-cytidine spacer were synthesized. They were designed to associate into a tetramer including an i-motif core built by intercalation of the C.C(+) pairs of the longer C stretch with the two dangling non-intercalated strands of the shorter C stretch at each end. Gel filtration chromatography shows that the non-intercalated C-rich ends give to this structure the capacity of forming extremely stable sms. Using C(7)GC(4) as a model, we find that the sms formation rate varies as the oligonucleotide concentration and increases at high temperature. Competitively with the tetramer involved in sms elongation, C(n)XC(m) oligonucleotides form i-motif dimers that compete with sms elongation. The dimer stability is strongly reduced when the pH is moved away from the cytidine pK. This results in an equilibrium shift towards the tetramer and in the acceleration of the sms formation rate. The chromatograms of the sms formed by C(7)GC(4) indicate a broad distribution. In a 1.5 mM solution incubated at 37 degrees C, the equilibrium distribution is centered on a molecular weight corresponding to the assembly of nine tetramers and the upper limit corresponds to 80 tetramers. The lifetime of this structure is about 4 days at 40 degrees C, pH 4.6.

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.

Self-assembly properties and dynamics of synthetic proteo-nucleic building blocks in solution and on surfaces.

Synthetic proteo-nucleic structures (PDNAs) encompassing a single-stranded DNA sequence covalently attached to a redox protein domain able to interact with surface or matrix were designed and characterized. They constitute versatile building blocks alternative to regular DNA for creating scaffolds with optical, electrical, or catalytic properties. PDNAs self-assemble in the presence of complementary oligonucleotides, to form a network of protein domains linked by double-stranded DNA segments. Electrophoretic and hydrodynamic behaviors of PDNAs and corresponding DNA were compared under electrophoresis and gel filtration conditions. Hybridization rates between small and large assemblies were characterized by rapid-mixing experiments. Results showed that the protein part significantly contributes to hydrodynamic behaviors of structures but marginally affects the conformation and hybridization properties of the nucleic domain. PDNA metal-mediated complexes with nitriloacetate-modified phospholipids can diffuse and interact at the surface of vesicles or supported membranes. Surface plasmon resonance analysis of membrane-PDNA interactions indicated that two protein units are required to allow stable surface association and that surface occupancy constrains assembly sizes. High-speed atomic force microscopy illustrated rapid lateral diffusion of assemblies on mica, revealing transient association between noncomplementary PDNA extremities and frequent trapping by surface defects. Regularly organized protein domains were visualized using a larger DNA framework.

Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase.

Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo- and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 A resolution crystal structure of a functionally competent yeast-human chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 A.

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry.

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimer's disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer's disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimer's disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-beta peptide (A beta) deposit. Two cases were apo epsilon 4/apo epsilon 3, and one case was apo epsilon 3/apoepsilon3. A known quantity of (13)C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 +/- 0.1 attomoles/microm(3) in the senile plaques and 2.2 +/- 0.49 attomoles/microm(3) in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 +/- 16 femtomol) is similar to the published quantity of A beta peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer's disease linked to the apo epsilon 4 allele, suggests new pathogenetic mechanisms.

Polarimetric surface plasmon resonance imaging biosensor.

We report the realization of a polarimetric surface plasmon resonance imaging system capable of dynamically resolving a change in the optical anisotropy of biochemical films. Anisotropies as small as 10(-3) refractive index unit on nanometer-thick samples can be resolved. As an example, we present here the dynamical anisotropy obtained by the electrical patterning of a film consisting of a self-assembled monolayer deposited on gold, covered with a phospholipid hemimembrane.

Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase.

NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures.

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.

The Sterolgene v0 cDNA microarray: a systemic approach to studies of cholesterol homeostasis and drug metabolism.

BACKGROUND: Cholesterol homeostasis and xenobiotic metabolism are complex biological processes, which are difficult to study with traditional methods. Deciphering complex regulation and response of these two processes to different factors is crucial also for understanding of disease development. Systems biology tools as are microarrays can importantly contribute to this knowledge and can also discover novel interactions between the two processes. RESULTS: We have developed a low density Sterolgene v0 cDNA microarray dedicated to studies of cholesterol homeostasis and drug metabolism in the mouse. To illustrate its performance, we have analyzed mouse liver samples from studies focused on regulation of cholesterol homeostasis and drug metabolism by diet, drugs and inflammation. We observed down-regulation of cholesterol biosynthesis during fasting and high-cholesterol diet and subsequent up-regulation by inflammation. Drug metabolism was down-regulated by fasting and inflammation, but up-regulated by phenobarbital treatment and high-cholesterol diet. Additionally, the performance of the Sterolgene v0 was compared to the two commercial high density microarray platforms: the Agilent cDNA (G4104A) and the Affymetrix MOE430A GeneChip. We hybridized identical RNA samples to the commercial microarrays and showed that the performance of Sterolgene is comparable to commercial arrays in terms of detection of changes in cholesterol homeostasis and drug metabolism. CONCLUSION: Using the Sterolgene v0 microarray we were able to detect important changes in cholesterol homeostasis and drug metabolism caused by diet, drugs and inflammation. Together with its next generations the Sterolgene microarrays represent original and dedicated tools enabling focused and cost effective studies of cholesterol homeostasis and drug metabolism. These microarrays have the potential of being further developed into screening or diagnostic tools.

CREM modulates the circadian expression of CYP51, HMGCR and cholesterogenesis in the liver.

We show for the first time that isoforms of the cAMP response element modulator Crem, regulate the circadian expression of Cyp51 and other cholesterogenic genes in the mouse liver. In the wild type mice the expression of Cyp51, Hmgs, Fpps, and Sqs is minimal between CT12 and CT16 and peaks between CT20 and CT24. Cyp51, Fpps, and Sqs lost the circadian behavior in Crem-/- livers while Hmgcr is phase advanced from CT20 to CT12. This coincides with a phase advance of lathosterol/cholesterol ratio, as detected by GC-MS. Overexpression of CREMtau and ICER has little effect on the Hmgcr proximal promoter while they influence expression from the CYP51 promoter. Our data indicate that Crem-dependent regulation of Cyp51 in the liver results in circadian expression of this gene. We propose that cAMP signaling might generally be involved in the circadian regulation of cholesterol synthesis on the periphery.