Cytochrome P450 Compound Research Articles

Insights into Cytochrome P450 Enzyme Catalyzed Defluorination of Aromatic Fluorides

The biodegradability of many fluorinated compounds is limited due to the robustness of the C‐F bond. Recently, experimental studies suggested the potential involvement of cytochrome P450 enzymes in facilitating aromatic defluorination, raising questions where this can be applied in biocatalysis. Our study offers an in‐depth computational examination into the oxidative defluorination process of a drug molecule, mediated by cytochrome P450 Compound I. We explored a large number of potential mechanisms, and identify two competitive low‐energy pathways that are initiated with an electrophilic attack on the aromatic ring, and followed by either a 1,2‐fluorine shift or a ring‐closure to form an epoxide intermediate. Both of these intermediates are shown to react rapidly through defluorination assisted by a solvent proton. Interestingly, defluorination in the vicinity of a heme group may generate a stable iron(III)‐fluoride complex, potentially leading to enzyme inactivation.

Insights into Cytochrome P450 Enzyme Catalyzed Defluorination of Aromatic Fluorides.

Density functional calculations establish a novel mechanism of aromatic defluorination by P450 Compound I. This is achieved via either an initial epoxide intermediate or through a 1,2-fluorine shift in an electrophilic intermediate, which highlights that the P450s can defluorinate fluoroarenes. However, in the absence of a proton donor a strong Fe-F bond can be obtained as shown from the calculations.

Substrate sulfoxidation by a biomimetic cytochrome P450 Compound I mimic: How do porphyrin and phthalocyanine equatorial ligands compare?

Substrate sulfoxidation by a biomimetic cytochrome P450 Compound I mimic: How do porphyrin and phthalocyanine equatorial ligands compare?

A three states model for hydrogen abstraction reactions with the cytochrome P450 compound I is revisited. Isolobal and isospin analogy among Fe(IV)=O, O = O and O

Radical abstraction reactions by the cytochrome P450 compound I are investigated on the basis of the one quartet two doublet model consisted of three configurations; 4{2[↑• +Por]–[↑• 3Fe(IV)=O •↑]} (4CpdI; S = 3/2), 2{2[↓• +Por]–[↑• 3Fe(IV)=O •↑]} (2CpdIa; S = 1/2) and 2{1[Por(↑↓)]–2[↑• Fe(V)=O]} (2CpdIb; S = 1/2) where Por denotes porphyrin and axial cysteine anion (–SR) is neglected. The dπ-pπ bond of 2CpdIb was highly spin-polarized in the transition-state region of hydrogen abstraction reactions, providing the doublet configuration; 2{1[Por(↑↓)]–2[↑↑•• Fe(IV)–O •↓]} (2CpdIb,oxyl). The oxyl-radical character of 2CpdIb,oxyl plays an important role for hydrogen radical abstraction from alkanes. The spin populations on the iron atom at the transition structures by available density functional theory (DFT) results were about 2.0 for 2CpdIb,oxyl, whereas they were about 1.0 for 4CpdI and 2CpdIa. On the other hand, the spin density population on the carbon atom of alkyl radical were negative and positive in sign for 2CpdIb,oxyl and 4CpdI, respectively, in consistent with their spin structures. In order to elucidate possible reaction pathways by the cytochrome P450 compound I, relative stabilities between 4CpdI and 2CpdIb were examined on the basis of available DFT and beyond DFT results. The refined DFT results obtained by the beyond DFT elucidated one of the possible reaction pathways for hydrogen abstractions; 2CpdIa → 2Cpdb → 2CpdIb,oxyl → 2(TS) → (rebound). Implications of the computational results are also discussed in relation to water oxidation in artificial catalysts.

In silico toxicity prediction, synthesis, characterization, antimicrobial and antioxidant activity of different di substituted chalcones

Right now the amalgamation of novel arrangement of 4'- chloro-3'- nitro-phenyl-(3-keto-1,2diene-1-flurobenzene), 4'- chloro-3'nitro-phenyl-(3-keto-1,2diene-1-chlorobenzene),4'- chloro-3'- nitro-phenyl-(3-keto-1,2diene-1-thiophene),4'- chloro-3'nitro-phenyl-(3-keto-1,2diene-1-pyridine), 4'- chloro-3'nitro-phenyl-(3-keto-1,2diene-1-furon).The target mixes were integrated by the Claisen-schimidt buildup utilizing corrosive and base as a response impetus. insilico enhancement strategies by utilization of various information base like molinspiration for atomic properties, bioactivity, ochem for organic information, pre ADME for pharmacokinetic properties, preToxicity for harmfulness profile of the mixes, swiss ADME for pharmacokinetic property, Swiss lethality for natural danger of the mixes, and Osiris(datawarrior) for poisonous quality forecast for dynamic lead particles ID. From the outcome mixes indented as dynamic leads they used to assess distinctive organic action. The mixes additionally finish the amees assessment for the most part for mutagenecity discovery that mixes shows mutagencity towards the test living being they likewise safe to hinder cytochrome P450 compound subunits like cyp1A2, cyp2C9, cyp2C19 which are situated in endoplasmic reticulum. Alpha beta unsaturated chalcones have fantastic cell reinforcement and antimicrobial action so the orchestrated mixes were screened for their antimicrobial by utilizing five bacterial strains and three parasitic strains and cancer prevention agent action by DPPH technique. From hostile to oxidant results compound 3a, 3b, 3c show critical action the measure esteems are closer to standard medication esteem. Antimicrobial movement results the compound 3a, 3c, 3e indicated noteworthy action against Bacillus subtilis, E Coli, Salmonella tyhphi, Pseudomonas aeruginosa though the compound 3b, 3d demonstrated moderate activitycompared to standard medication vale. Compound 3b, 3d demonstrated fantastic antifungal action against Candida albicans, Aspergillusniger, Alternariaalternata. The mixes 3a, 3c, 3e show moderate antifungal movement when contrasted with standard worth.

Characterization of a selenocysteine-ligated P450 compound I reveals direct link between electron donation and reactivity.

Strong electron-donation from the axial thiolate ligand of cytochrome P450 has been proposed to increase the reactivity of compound I with respect to C-H bond activation. However, it has proven difficult to test this hypothesis, and a direct link between reactivity and electron donation has yet to be established. To make this connection, we have prepared a selenolate-ligated cytochrome P450 compound I intermediate. This isoelectronic perturbation allows for direct comparisons with the wild-type enzyme. Selenium incorporation was achieved using a cysteine auxotrophic Escherichia coli strain. The intermediate was prepared with meta-chloroperbenzoic acid and characterized by UV-visible, Mössbauer and electron paramagnetic resonance spectroscopies. Measurements revealed increased asymmetry around the ferryl moiety, consistent with increased electron donation from the axial selenolate ligand. In line with this observation, we find that the selenolate-ligated compound I cleaves C-H bonds more rapidly than the wild-type intermediate.

Oxygen Atom Transfer Using an Iron(IV)-Oxo Embedded in a Tetracyclic N-Heterocyclic Carbene System: How Does the Reactivity Compare to Cytochrome P450 Compound I?

N-Heterocyclic carbenes (NHC) are commonly featured as ligands in transition metal catalysis. Recently, a cyclic system containing four NHC groups with a central iron atom was synthesized and its iron(IV)-oxo species, [FeIV (O)(cNHC4 )]2+ , was characterized. This tetracyclic NHC ligand system may give the iron(IV)-oxo species unique catalytic properties as compared to traditional non-heme and heme iron ligand systems. Therefore, we performed a computational study on the structure and reactivity of the [FeIV (O)(cNHC4 )]2+ complex in substrate hydroxylation and epoxidation reactions. The reactivity patterns are compared with cytochrome P450 Compound I and non-heme iron(IV)-oxo models and it is shown that the [FeIV (O)(cNHC4 )]2+ system is an effective oxidant with oxidative power analogous to P450 Compound I. Unfortunately, in polar solvents, a solvent molecule will bind to the sixth ligand position and decrease the catalytic activity of the oxidant. A molecular orbital and valence bond analysis provides insight into the origin of the reactivity differences and makes predictions of how to further exploit these systems in chemical catalysis.

Will 1,2-dihydro-1,2-azaborine-based drugs resist metabolism by cytochrome P450 compound I?

1,2-dihydro-1,2-azaborine is a structural and electronic analogue of benzene which is able to occupy benzene-binding pockets in T4 lysozyme and has been proposed as suitable arene-mimicking group for biological and pharmaceutical applications. Its applicability in a biological context requires it to be able to resist modification by xenobiotic-degrading enzymes like the P450 cytochromes. Quantum chemical computations described in this work show that 1,2-dihydro-1,2-azaborine is much more prone to modification by these enzymes than benzene, unless steric crowding of the ring prevents it from reaching the active site, or otherwise only allows reaction at the less reactive C4-position. This novel heterocyclic compound is therefore expected to be of limited usefulness as an aryl bioisostere.

Origin of the Enhanced Reactivity of μ-Nitrido-Bridged Diiron(IV)-Oxo Porphyrinoid Complexes over Cytochrome P450 Compound I

μ-Nitrido-bridged diiron porphyrins and phthalocyanines are known to be efficient oxidants that are able to oxidize some of the strongest C–H bonds in nature, such as the one in methane. The origin of their catalytic efficiency is poorly understood, and in order to gain insight into the structural and electronic features of this chemical system, we performed a detailed and systematic study into their chemical properties and reactivities using density functional theory. Our work shows that μ-nitrido-bridged diiron porphyrins and phthalocyanines are highly active catalytic oxidants, which react with methane with very low reaction barriers and a rate-determining hydrogen-atom-abstraction step. Furthermore, the μ-nitrido-bridged diiron porphyrin and phthalocyanine complexes react with a free energy of activation that is more than 10 kcal mol–1 lower in energy than that found for cytochrome P450 Compound I, which is known to be one of the most efficient C–H hydroxylating agents in Nature. We have analyzed the ...

Decarboxylation of fatty acids to terminal alkenes by cytochrome P450 compound I.

OleT(JE), a cytochrome P450, catalyzes the conversion of fatty acids to terminal alkenes using hydrogen peroxide as a cosubstrate. Analytical studies with an eicosanoic acid substrate show that the enzyme predominantly generates nonadecene and that carbon dioxide is the one carbon coproduct of the reaction. The addition of hydrogen peroxide to a deuterated substrate-enzyme (E-S) complex results in the transient formation of an iron(IV) oxo π cation radical (Compound I) intermediate which is spectroscopically indistinguishable from those that perform oxygen insertion chemistries. A kinetic isotope effect for Compound I decay suggests that it abstracts a substrate hydrogen atom to initiate fatty acid decarboxylation. Together, these results indicate that the initial mechanism for alkene formation, which does not result from oxygen rebound, is similar to that widely suggested for P450 monooxygenation reactions.

An attempt to evaluate the effect of proton-coupled electron transfer on the H-abstraction step of the reaction between 1,1-dimethylhydrazine and cytochrome P450 compound I

A series of density functional theory and G4 calculations are performed to quantify the extent to which proton-coupled electron transfer (PCET) lowers the barrier for H-abstraction in the reaction between 1,1-dimethylhydrazine (UDMH) and the compound I intermediate of a cytochrome P450 enzyme. The homolytic bond dissociation energy of UDMH is larger than that of 1,4-cyclohexadiene; nevertheless, the H-abstraction barrier is significantly lower for the former substrate. The barrier lowering in the UDMH reaction caused by PCET is roughly estimated to be 9kcal/mol. Valence bond structures are analyzed in detail to figure out how PCET lowers the barrier height.

Cytochrome P450 compound I in the plane wave pseudopotential framework: GGA electronic and geometric structure of thiolate-ligated iron(IV)-oxo porphyrin.

The cytochromes P450 constitute a ubiquitous family of metalloenzymes, catalyzing manifold reactions of biological and synthetic importance via a thiolate-ligated iron-oxo (IV) porphyrin radical species denoted compound I (Cpd I). Experimental investigations have implicated this intermediate in a broad spectrum of biophysically interesting phenomena, further augmenting the importance of a Cpd I model system. Ab initio molecular dynamics, including Car-Parrinello and path integral methods, conjoin electronic structure theory with finite temperature simulation, affording tools most valuable to approach such enzymes. These methods are typically driven by density functional theory (DFT) in a plane-wave pseudopotential framework; however, existing studies of Cpd I have been restricted to localized Gaussian basis sets. The appropriate choice of density functional and pseudopotential for such simulations is accordingly not obvious. To remedy this situation, a systematic benchmarking of thiolate-ligated Cpd I is performed using several generalized-gradient approximation (GGA) functionals in the Martins-Troullier and Vanderbilt ultrasoft pseudopotential schemes. The resultant electronic and structural parameters are compared to localized-basis DFT calculations using GGA and hybrid density functionals. The merits and demerits of each scheme are presented in the context of reproducing existing experimental and theoretical results for Cpd I.

Expression of Fusion Proteins of Aspergillus terreus Reveals a Novel Allene Oxide Synthase

Aspergilli oxidize C18 unsaturated fatty acids by dioxygenase-cytochrome P450 fusion proteins to signal molecules involved in reproduction and host-pathogen interactions. Aspergillus terreus expresses linoleate 9R-dioxygenase (9R-DOX) and allene oxide synthase (AOS) activities in membrane fractions. The genome contains five genes (ATEG), which may code for a 9R-DOX-AOS fusion protein. The genes were cloned and expressed, but none of them oxidized 18:2n-6 to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE). ATEG_02036 transformed 9R-HPODE to an unstable allene oxide, 9(R),10-epoxy-10,12(Z)-octadecadienoic acid. A substitution in the P450 domain (C1073S) abolished AOS activity. The N964V and N964D mutants both showed markedly reduced AOS activity, suggesting that Asn(964) may facilitate homolytic cleavage of the dioxygen bond of 9R-HPODE with formation of compound II in analogy with plant AOS (CYP74) and prostacyclin synthase (CYP8A1). ATEG_03992 was identified as 5,8-linoleate diol synthase (5,8-LDS). Replacement of Asn(878) in 5,8-LDS with leucine (N878L) mainly shifted ferryl oxygen insertion from C-5 toward C-6, but replacements of Gln(881) markedly affected catalysis. The Q881L mutant virtually abolished the diol synthase activity. Replacement of Gln(881) with Asn, Glu, Asp, or Lys residues augmented the homolytic cleavage of 8R-HPODE with formation of 10-hydroxy-8(9)-epoxy-12(Z)-octadecenoic acid (erythro/threo, 1-4:1) and/or shifted ferryl oxygen insertion from C-5 toward C-11. We conclude that homolysis and heterolysis of the dioxygen bond with formation of compound II in AOS and compound I in 5,8-LDS are influenced by Asn and Gln residues, respectively, of the I-helices. AOS of A. terreus appears to have evolved independently of CYP74 but with an analogous reaction mechanism.

Optimized Experimental Design for the Estimation of Enzyme Kinetic Parameters: An Experimental Evaluation

A set of compounds (n = 30), including traditional cytochrome P450 substrates and compounds from AstraZeneca's compound library, was used in an experimental evaluation of an optimal design approach (ODA) for the estimation of enzyme kinetic parameters (CL(int), V(max), and K(m)). A depletion method previously shown to provide reliable results, the multiple depletion curves method (MDCM), was used as reference. Experiments were conducted with human liver microsomes, and samples were analyzed using liquid chromatography-tandem mass spectrometry. CL(int) estimated with the ODA were in >90% of the cases within a 2-fold difference compared with MDCM estimates. In addition, good agreement was generally seen for V(max) and K(m) estimates between the two methods as >80% of the estimates were within or almost within a 2-fold difference. The variability in V(max) and K(m) estimates were generally higher than for CL(int) estimates. In addition, decreased substrate turnover considerably increased the variability in V(max) and K(m) estimates, whereas only a modest increase was observed for CL(int) estimates. The experimental design of using multiple starting concentrations for the estimation of enzyme kinetics was shown to be appropriate even when there was a limitation to the number of samples. The method allowed for good estimates of CL(int) and also for V(max) and K(m) in many cases. Hence, this approach is a good alternative for the estimation of enzyme kinetic parameters, especially if enzyme saturation and an assessment of a potential risk for nonlinear metabolism are of interest.

Zinc-Substituted Cytochrome P450cam: Characterization of Protein Conformers F420 and F450 by Photoinduced Electron Transfer

Metal substitution of heme proteins is widely applied in the study of biologically relevant electron transfer (ET) reactions. It has been shown that many modified proteins remain in their native conformation and can provide useful insights into the molecular mechanism of electron transfer between the native protein and its substrates. We investigated ET reactions between zinc-substituted cytochrome P450(cam) and small organic compounds such as quinones and ferrocene, which are capable of accessing the protein's hydrophobic channel and binding close to the active site, like its native substrate, camphor. Following the substitution method developed by Gunsalus and co-workers [Wagner, G. C., et al. (1981) J. Biol. Chem. 256, 6262-6265], we have identified two dominant forms of the zinc-substituted protein, F450 and F420, that exhibit different photophysical and photochemical properties. The ET behavior of F420 suggests that hydrophobic redox-active ligands are able to penetrate the hydrophobic channel and place themselves in the direct vicinity of the Zn-porphyrin. In contrast, the slower ET quenching rates observed in the case of F450 indicate that the association is weak and occurs outside of the protein channel. Therefore, we conclude that F420 corresponds to the open structure of the native cytochrome P450(cam) while F450 has a closed or partially closed channel that is characteristic of the camphor-containing cytochrome P450(cam). The existence of two distinct conformers of Zn-bound P450(cam) is consistent with the findings of Goodin and co-workers [Lee, Y.-T., et al. (2010) Biochemistry 49, 3412-3419] and has significant consequences for future electron transfer studies on this popular metalloenzyme.

Rate-Controlling Isomerizations in Fatty Acid Oxidations by a Cytochrome P450 Compound I

KIE events: Hydroxylations of fatty acids by cytochrome P450 119 Compound I display no intermolecular kinetic isotope effect (KIE) in buffer and increase in rate with increasing chain length (see graph). With glycerol, the rate of reaction of lauric acid increases, and the KIE is revealed. The mechanism involves reversible formation of an unreactive complex of a fatty acid with Compound I and its isomerization to a reactive one.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone

The genes for polyketide synthases (PKSs), enzymes that assemble the carbon backbones of many secondary metabolites, often cluster with other secondary pathway genes. We describe here the first lichen PKS cluster likely to be implicated in the biosynthesis of a depside and a depsidone, compounds in a class almost exclusively produced by lichen fungi (mycobionts). With degenerate PCR with primers biased toward presumed PKS genes for depsides and depsidones we identified among the many PKS genes in Cladonia grayi four (CgrPKS13-16) potentially responsible for grayanic acid (GRA), the orcinol depsidone characteristic of this lichen. To single out a likely GRA PKS we compared mRNA and GRA induction in mycobiont cultures using the four candidate PKS genes plus three controls; only CgrPKS16 expression closely matched GRA induction. CgrPKS16 protein domains were compatible with orcinol depside biosynthesis. Phylogenetically CgrPKS16 fell in a new subclade of fungal PKSs uniquely producing orcinol compounds. In the C. grayi genome CgrPKS16 clustered with a CytP450 and an o-methyltransferase gene, appropriately matching the three compounds in the GRA pathway. Induction, domain organization, phylogeny and cluster pathway correspondence independently indicated that the CgrPKS16 cluster is most likely responsible for GRA biosynthesis. Specifically we propose that (i) a single PKS synthesizes two aromatic rings and links them into a depside, (ii) the depside to depsidone transition requires only a cytochrome P450 and (iii) lichen compounds evolved early in the radiation of filamentous fungi.

A mononuclear nonheme iron(iv)-oxo complex which is more reactive than cytochrome P450 model compound I

A highly reactive mononuclear nonheme iron(IV)-oxo complex with a low-spin (S = 1) triplet ground state in both C–H bond activation and oxo transfer reactions is reported; this nonheme iron(IV)-oxo complex is more reactive than an iron(IV)-oxo porphyrin π-cation radical (i.e., a model of cytochrome P450 compound I) and is the most reactive species in kinetic studies among nonheme iron(IV)-oxo complexes reported so far. DFT calculations support the experimental results with extremely low activation barriers in the C–H bond activation of cyclohexane and 1,4-cyclohexadiene. The DFT calculations reveal that the S = 1 state is set up to easily lead to the highly reactive S = 2 high-spin iron(IV)-oxo species.

Cytochrome P450 Compound I: Capture, Characterization, and C-H Bond Activation Kinetics

Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) ~ 100 kilocalories per mole], with an apparent second-order rate constant of k(app) = 1.1 × 10(7) per molar per second at 4°C. Direct measurements put a lower limit of k ≥ 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second.