Human P450 1B1 Research Articles

Oxidation of Naringenin, Apigenin, and Genistein by Human Family 1 Cytochrome P450 Enzymes and Comparison of Interaction of Apigenin with Human P450 1B1.1 and Scutellaria P450 82D.1.

Naringenin, an initial synthesized flavanone in various plant species, is further utilized for production of many biologically active flavonoids, e.g., apigenin, eriodictyol, and genistein, by various plant enzymes including cytochrome P450s (P450s or CYPs). We examined how these flavonoids are oxidized by human P450 family 1 and 2A enzymes. Naringenin was principally oxidized at the 3'-position to form eriodictyol by CYP1 enzymes more efficiently than by CYP2A enzymes, and the resulting eriodictyol was further oxidized to two penta-hydroxylated products. In contrast to plant P450 enzymes, these human P450s did not mediate the desaturation of naringenin and eriodictyol to give apigenin and luteolin, respectively. Apigenin was oxidized at the C3' and C6 positions to form luteolin and scutellarein by these P450s. CYP1B1.1 and 1B1.3 had high activities in apigenin 6-hydroxylation with a homotropic cooperative manner, as has been observed previously in chrysin 6-hydroxylation (Nagayoshi et al., Chem. Res. Toxicol. 2019, 32, 1268-1280). Molecular docking analysis suggested that CYP1B1 had two apigenin binding sites and showed similarities in substrate recognition sites to plant CYP82D.1, one of the enzymes in catalyzing apigenin and chrysin 6-hydroxylations in Scutellaria baicalensis. The present results suggest that human CYP1 enzymes and CYP2A13 in some reactions have important roles in the oxidation of naringenin, eriodictyol, apigenin, and genistein and that human CYP1B1 and Scutellaria CYP82D.1 have similarities in their SRS regions, catalyzing 6-hydroxylation of both apigenin and chrysin.

Oxidation of 3’-methoxyflavone, 4’-methoxyflavone, and 3’,4’-dimethoxyflavone and their derivatives having 5,7-dihydroxyl moieties by human cytochromes P450 1B1 and 2A13

Oxidation of 3’-methoxyflavone, 4’-methoxyflavone, and 3’,4’-dimethoxyflavone and their derivatives containing 5,7-dihydroxyl groups by human cytochrome P450 (P450 or CYP) 1B1 and 2A13 was determined using LC-MS/MS systems. 3’-Methoxyflavone and 4’-methoxyflavone were mainly O-demethylated to form 3’-hydroxyflavone and 4’-hydroxyflavone, respectively, and then 3’,4’-dihydroxyflavone at higher rates with CYP1B1 than with CYP2A13. 4’-Methoxy-5,7-dihydroxyflavone (acacetin) was found to be demethylated by CYP1B1 and 2A13 to form 4’,5,7-trihydroxyflavone (apigenin) at rates of 0.098−1 and 0.42 min−1, respectively. 3’-Methoxy-5,7-dihydroxyflavone was also demethylated by both P450s, with CYP2A13 being more active. 3’,4’-Dimethoxyflavone was a good substrate for CYP1B1 but not for CYP2A13 and was found to be mainly O-demethylated to form 3’,4’-dihydroxyflavone (at a rate of 4.2 min−1) and also several ring-oxygenated products having m/z 299 fragments. Molecular docking analysis supported the proper orientation for formation of these products by CYP1B1. Our present results showed that 3’- and 4’-methoxyflavone can be oxidised to their O-demethylated products and, to a lesser extent, to ring oxidation products by both P450s 1B1 and 2A13 and that 3’,4’-dimethoxyflavone is a good substrate for CYP1B1 in forming both O-demethylated and ring-oxidation products. Introduction of a 57diOHF moiety into these methoxylated flavonoids caused decreased in oxidation by CYP1B1 and 2A13.

Halogen‐π Interactions in the Cytochrome P450 Active Site: Structural Insights into Human P450 2B6 Substrate Selectivity.

Numerous CYP2B6 substrates including drugs and environmental chemicals are halogenated. To assess the role of halogen‐π bonds in substrate selectivity and orientation in the active site, structures of four CYP2B6 monoterpenoid complexes were solved by X‐ray crystallography. Bornyl bromide exhibited dual orientations in the active site with the predominant orientation revealing a bromine‐π bond with the Phe‐108 side chain. Bornane demonstrated two orientations with equal occupancy; in both the C2‐atom that bears the bromine in bornyl bromide was displaced by more than 2.5 Å compared with the latter complex. The bromine in myrtenyl bromide π‐bonded with Phe‐297 in CYP2B6, whereas the two major orientations in the active site mutant I114V exhibited bromine‐π interactions with two additional residues, Phe‐108 and Phe‐115. Analysis of existing structures suggests that halogen‐π interactions may be unique to the CYP2B enzymes within CYP family 2 but are also important for CYP3A enzymes.Support or Funding InformationES003619 to Dr. James R. Halpert

Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with Ks values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute-1) than CYP2A13 (∼0.02 minute-1) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute-1) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute-1) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute-1). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences.

Structural and thermodynamic basis of (+)-α-pinene binding to human cytochrome P450 2B6.

Despite recent advances in atomic-level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-α-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-α-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-α-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme-ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility.

X‐ray Crystal Structure of the Cytochrome P450 2B4 Active Site Mutant F297A in Complex with Clopidogrel: Insights into Compensatory Rearrangements of the Binding Pocket

Prior X‐ray crystal structures of rabbit cytochrome P450 2B4 and human P450 2B6 have revealed the pivotal role of side‐chain rearrangement of active site residues F206 and F297 in accommodating various inhibitors or substrates. To explore further the structural basis of ligand binding, the crystal structure of 2B4 F297A was solved in complex with the drug clopidogrel. The structure demonstrated the reorientation of ligand in the active site such that the acetoxy group is oriented toward the heme, whereas the thiophene moiety now extends to the void region of A297 on the I‐helix. Interestingly, movement of the I‐helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A‐virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the F297A structure or a virtual mutant where the I‐helix was also repositioned, oriented clopidogrel preferentially with either the acetoxy or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate the substrate in a distinct orientation and has important implications for understanding the role of active site residues in functional differences among related P450 enzymes. Supported by NIH grant ES03619.

Binding of Diverse Environmental Chemicals with Human Cytochromes P450 2A13, 2A6, and 1B1 and Enzyme Inhibition

A total of 68 chemicals including derivatives of naphthalene, phenanthrene, fluoranthene, pyrene, biphenyl, and flavone were examined for their abilities to interact with human P450s 2A13 and 2A6. Fifty-one of these 68 chemicals induced stronger Type I binding spectra (iron low- to high-spin state shift) with P450 2A13 than those seen with P450 2A6, i.e., the spectral binding intensities (ΔAmax/Ks ratio) determined with these chemicals were always higher for P450 2A13. In addition, benzo[c]phenanthrene, fluoranthene, 2,3-dihydroxy-2,3-dihydrofluoranthene, pyrene, 1-hydroxypyrene, 1-nitropyrene, 1-acetylpyrene, 2-acetylpyrene, 2,5,2',5'-tetrachlorobiphenyl, 7-hydroxyflavone, chrysin, and galangin were found to induce a Type I spectral change only with P450 2A13. Coumarin 7-hydroxylation, catalyzed by P450 2A13, was strongly inhibited by 2'-methoxy-5,7-dihydroxyflavone, 2-ethynylnaphthalene, 2'-methoxyflavone, 2-naphththalene propargyl ether, acenaphthene, acenaphthylene, naphthalene, 1-acetylpyrene, flavanone, chrysin, 3-ethynylphenanthrene, flavone, and 7-hydroxyflavone; these chemicals induced Type I spectral changes with low Ks values. On the basis of the intensities of the spectral changes and inhibition of P450 2A13, we classified the 68 chemicals into eight groups based on the order of affinities for these chemicals and inhibition of P450 2A13. The metabolism of chemicals by P450 2A13 during the assays explained why some of the chemicals that bound well were poor inhibitors of P450 2A13. Finally, we compared the 68 chemicals for their abilities to induce Type I spectral changes of P450 2A13 with the Reverse Type I binding spectra observed with P450 1B1: 45 chemicals interacted with both P450s 2A13 and 1B1, indicating that the two enzymes have some similarty of structural features regarding these chemicals. Molecular docking analyses suggest similarities at the active sites of these P450 enzymes. These results indicate that P450 2A13, as well as Family 1 P450 enzymes, is able to catalyze many detoxication and activation reactions with chemicals of environmental interest.

Increased mutant frequency in V79 cells expressing human CYP P450 1B1

Increased mutant frequency in V79 cells expressing human CYP P450 1B1

Functional Characterization of Cytochromes P450 2B from Woodrats

Much of the structure‐function information on cytochromes P450 (P450) 2B is from a limited number of pharmacologically relevant species. Recent data suggest that desert woodrats (Neotoma lepida) have multiple P450 2B enzymes with greater than 85% identity to P450 2B1. Populations of N. lepida consuming creosote (Larrea tridentata) or juniper (Juniperus osteosperma) have differential expression of P450s 2B. Following previous protocols, we modified, expressed, and purified one P450 2B from one woodrat fed a creosote diet and one fed a juniper diet. (−)‐a‐pinene, β‐myrcene, and d‐limonene, which are common in juniper and previously reported to be inhibitors of P450 2B1, show very high affinity (<50 nM) for both woodrat enzymes. The woodrat enzymes show similar activity to P450 2B1 with 7‐ethoxy‐4‐ trifluoromethyl‐coumarin (7‐EFC), but significant differences in activity and product profile are seen in ticlopidine metabolism. Residue 262 shows a lysine – arginine difference between the two enzymes. This residue has demonstrated functional differences in the human P450 2B6, and the known human genetic polymorphism K262R produces significant changes in ligand interactions. Differences in P450 2B structure in woodrats may confer functional differences in the metabolism of dietary toxins. (Supported by ES003619 and T32‐DK07233).

Dual Ligand Complexes of Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4 with Amlodipine Reveal Substrate Access Channels into the Active Site

Substrate access channels have been predicted previously for several mammalian P450 enzymes based mainly on molecular dynamics simulations. We now report X‐ray crystal structures of human P450 2B6 and rabbit P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine. The presence of the two drug molecules identifies clear access pathways in each P450. Based on the access channel nomenclature, 2f and 2a channels pointing towards the membrane correlate well with amlodipine access into the active site of P450 2B4. However, amlodipine appears to enter the active site of human P450 2B6 only via channel 2f. The results also indicate that key arginine residues located on the surface and at the entrance of the substrate access channels in each of the above P450s may play a crucial role in guiding substrate entry. In addition, secondary structure elements B′, F, F′, G′ and part of helix G were substantially retracted compared with the 4‐(4‐chlorophenyl)imidazole complexes of 2B4 and 2B6. Conformational variations were predominantly localized to the above elements that surround the substrate access channels as seen previously in the open P450cam structure. To our knowledge, the structures represent the first clear evidence from X‐ray crystallography of the substrate access channels into the active site in mammalian P450s. Supported by NIH grant ES03619.

Functional analysis of cytochrome P450 2B isoforms using ticlopidine as an active site probe

A recent study of in vitro metabolism of ticlopidine by reconstituted human P450 2B6 and rabbit P450 2B4 indicated that both enzymes generated multiple and similar metabolites but in different ratios. In this study, we determined the metabolism of ticlopidine by additional cytochrome P450 2B enzymes, including rat P450 2B1 and dog P450 2B11. Based on the X‐ray crystal structure of the P450 2B4‐ticlopidine complex, two active site mutants, F206A and F297A were also created. All enzymes were expressed in E. coli as N‐terminal modified, and C‐terminal His‐tagged constructs termed 2B4dH. The separation and characterization of the different metabolites of ticlopidine were achieved using HPLC‐MS/MS. P450 2B1 and 2B11 produced ticlopidine N‐oxide as the major product, and the production of this metabolite by the P450 2B subfamily exhibited a rank order of 2B1 > 2B11 > 2B4 > 2B6. The pattern of the products observed for P450 2B1 and 2B11 was similar to that of P450 2B4. In contrast, the 2B4dH F206A and F297A mutants formed predominantly ticlopidine S‐oxide dimer previously seen with P450 2B6. The highest rate of the dimer formation was observed in F206A compared with other 2B isoforms including 2B4dH F297A. These results suggest that ticlopidine is a valuable probe for differentiating P450 2B subfamily members and for assessing the effect of site‐directed mutations.(Supported by NIH grant ES003619)

Spectral Modification and Catalytic Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2A6, and 2A13 by Four Chemopreventive Organoselenium Compounds

Several organoselenium compounds including benzyl selenocyanate (BSC), 1,2-phenylenebis(methylene)selenocyanate (o-XSC), 1,3-phenylenebis(methylene)selenocyanate (m-XSC), and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) have been shown to prevent cancers caused by polycyclic aromatic hydrocarbons (PAHs) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in experimental animals; these chemical carcinogens are activated by human P450 1 and 2A family enzymes, respectively, to carcinogenic metabolites. In this study, we examined whether these selenium compounds interact with and inhibit human P450 1 and 2A enzymes in vitro. Four organoselenium compounds induced reverse Type I binding spectra with P450 1A1, 1A2, and 1B1 and Type I binding spectra with P450 2A6 and 2A13. The spectral dissociation constants (K(s)) for the interaction of P450 1B1 with these chemicals were 3.6-5.7 μM; the values were lower than those with seen with P450 1A1 (19-30 μM) or 1A2 (6.3-13 μM). The K(s) values for Type I binding of P450 2A13 with m-XSC and BSC were both 0.20 μM; the values were very low compared to those for the interaction of P450 2A6 with m-XSC (5.7 μM) and BSC (2.0 μM). Four selenium compounds directly inhibited 7-ethoxyresorufin O-deethylation activities catalyzed by P450 1A1, 1A2, and 1B1 with IC(50) values <1.0 μM, except for the inhibition of P450 1A2 by BSC (1.3 μM). Coumarin 7-hydroxylation activities of P450 2A13 were more inhibited by four selenium compounds than those of P450 2A6, with IC(50) values of 0.22-1.4 μM for P450 2A13 and 2.4-6.2 μM for P450 2A6. Molecular docking studies of the interaction of four organoselenium compounds with human P450 enzymes suggest that these chemicals can be docked into the active sites of these human P450 enzymes and that the sites of the selenocyanate functional groups of these chemicals differ between the P450 1 and 2A family enzymes.

Structural Characterization of the Complex between α-Naphthoflavone and Human Cytochrome P450 1B1

The atomic structure of human P450 1B1 was determined by x-ray crystallography to 2.7 Å resolution with α-naphthoflavone (ANF) bound in the active site cavity. Although the amino acid sequences of human P450s 1B1 and 1A2 have diverged significantly, both enzymes exhibit narrow active site cavities, which underlie similarities in their substrate profiles. Helix I residues adopt a relatively flat conformation in both enzymes, and a characteristic distortion of helix F places Phe(231) in 1B1 and Phe(226) in 1A2 in similar positions for π-π stacking with ANF. ANF binds in a distinctly different orientation in P450 1B1 from that observed for 1A2. This reflects, in part, divergent conformations of the helix B'-C loop that are stabilized by different hydrogen-bonding interactions in the two enzymes. Additionally, differences between the two enzymes for other amino acids that line the edges of the cavity contribute to distinct orientations of ANF in the two active sites. Thus, the narrow cavity is conserved in both P450 subfamily 1A and P450 subfamily 1B with sequence divergence around the edges of the cavity that modify substrate and inhibitor binding. The conservation of these P450 1B1 active site amino acid residues across vertebrate species suggests that these structural features are conserved.

Structure−Function Relationships of Inhibition of Human Cytochromes P450 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 Flavonoid Derivatives

Structure-function relationships for the inhibition of human cytochrome P450s (P450s) 1A1, 1A2, 1B1, 2C9, and 3A4 by 33 flavonoid derivatives were studied. Thirty-two of the 33 flavonoids tested produced reverse type I binding spectra with P450 1B1, and the potencies of binding were correlated with the abilities to inhibit 7-ethoxyresorufin O-deethylation activity. The presence of a hydroxyl group in flavones, for example, 3-, 5-, and 7-monohydroxy- and 5,7-dihydroxyflavone, decreased the 50% inhibition concentration (IC50) of P450 1B1 from 0.6 μM to 0.09, 0.21, 0.25, and 0.27 μM, respectively, and 3,5,7-trihydroxyflavone (galangin) was the most potent, with an IC50 of 0.003 μM. The introduction of a 4'-methoxy- or 3',4'-dimethoxy group into 5,7-dihydroxyflavone yielded other active inhibitors of P450 1B1 with IC50 values of 0.014 and 0.019 μM, respectively. The above hydroxyl and/or methoxy groups in flavone molecules also increased the inhibition activity with P450 1A1 but not always toward P450 1A2, where 3-, 5-, or 7-hydroxyflavone and 4'-methoxy-5,7-dihydroxyflavone were less inhibitory than flavone itself. P450 2C9 was more inhibited by 7-hydroxy-, 5,7-dihydroxy-, and 3,5,7-trihydroxyflavones than by flavone but was weakly inhibited by 3- and 5-hydroxyflavone. Flavone and several other flavonoids produced type I binding spectra with P450 3A4, but such binding was not always related to the inhibitiory activities toward P450 3A4. These results indicate that there are different mechanisms of inhibition for P450s 1A1, 1A2, 1B1, 2C9, and 3A4 by various flavonoid derivatives and that the number and position of hydroxyl and/or methoxy groups highly influence the inhibitory actions of flavonoids toward these enzymes. Molecular docking studies suggest that there are different mechanisms involved in the interaction of various flavonoids with the active site of P450s, thus causing differences in inhibition of these P450 catalytic activities by flavonoids.

Structural importance of residue 334 in the stability of cytochromes P450 2B6 and 2B11

Human P450 2B6 and canine P450 2B11 play an important role in xenobiotic metabolism, bioremediation research, and activation of anti‐cancer prodrugs. Thorough biochemical and biophysical characterization of the enzymes has been impeded by relatively low stability compared to the extensively studied rat 2B1 and rabbit 2B4. Comparison of the amino acid sequences of N‐terminal truncated and C‐terminal His‐tagged constructs revealed seven sites that are conserved between the more stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. Site‐directed mutagenesis at these positions showed that P334→ Ser substitution increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure‐perturbation spectroscopy. Compressibility of the heme pocket in the four P450 2B enzymes and their P334S (2B6 and 2B11) or S334P (2B1 and 2B4) mutants was assessed from the pressure induced displacement of the Soret band. The variants of all four enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterparts. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Mutation of the non‐active site residue P334→ Ser could find applications in drug metabolism and gene therapy for cancer treatment.

Functional and conformational modulation of human cytochrome P450 1B1 by anionic phospholipids

We investigated the interaction of human P450 1B1 (CYP1B1) with various phospholipid bilayers using the N-terminally deleted (Delta2-4)CYP1B1 and (Delta2-26)CYP1B1 enzymes. Among anionic phospholipids, phosphatidic acid (PA) and cardiolipin specifically increased the catalytic activities, membrane binding affinities, and thermal stabilities of both CYP1B1 proteins when phosphatidylcholine matrix was gradually replaced with these anionic phospholipids. PA- or cardiolipin-dependent changes of CYP1B1 conformation were revealed by altered Trp fluorescence and CD spectra. However, both PA and cardiolipin exerted more significant effects with the (Delta2-4)CYP1B1 than the (Delta2-26)CYP1B1 implying the functional importance of N-terminal region for the interaction with the phospholipid membranes. In contrast, other anionic phospholipids such as phosphatidylserine and the neutral phospholipid phosphatidylethanolamine had no apparent effects on the catalytic activity or conformation of CYP1B1. These data suggest that the chemical and physical properties of membranes influenced by PA or cardiolipin composition are critical for the functional roles of CYP1B1.

Investigation of the Mechanisms Underlying the Differential Effects of the K262R Mutation of P450 2B6 on Catalytic Activity

Human P450 2B6 is a polymorphic enzyme involved in the oxidative metabolism of a number of clinically relevant substrates. The lysine 262-to-arginine mutant of cytochrome P450 2B6 (P450 2B6.4) has been shown to have differential effects on P450 2B6 catalytic activity. We reported previously that the mutant enzyme was unable to metabolize 17-alpha-ethynylestradiol (17EE) or become inactivated by 17EE or efavirenz, which are inactivators of the wild-type enzyme. Studies were performed to elucidate the mechanism by which this mutation affects P450 2B6 catalytic activity. Studies using phenyldiazene to investigate differences between the active site topologies of the wild-type and mutant enzymes revealed only minor differences. Likewise, Ks values for the binding of both benzphetamine and efavirenz were comparable between the two enzymes. Using the alternate oxidant tert-butyl hydroperoxide, the mutant enzyme was inactivated by both 17EE and efavirenz. The stoichiometry of 17EE and efavirenz metabolism by P450s 2B6 and 2B6.4 revealed that the mutant enzyme was more uncoupled, producing hydrogen peroxide as the primary product. Interestingly, the addition of cytochrome b5 improved the coupling of the mutant, resulting in increased catalytic activity. In the presence of cytochrome b5 the variant readily metabolized 17EE and was inactivated by both 17EE and efavirenz. It is therefore proposed that the oxyferrous or iron-peroxo intermediate formed by the mutant enzyme in the presence of 17EE and efavirenz may be less stable than the same intermediates formed by the wild-type enzyme.

Engineering of Human Cytochrome P450 2B6 for Enhanced Expression, Stability, and Functional Studies of the Wild‐Type and Genetic Variants

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism and increasing number of genetic variants, thorough biochemical and biophysical characterization has been impeded due to low expression and stability. Recently, an N‐terminal modified 2B6 (2B6dH) mutant was engineered termed L264F, which showed enhanced expression and stability and retained functional integrity compared with 2B6dH. To further enhance the expression and stability of 2B6dH construction is underway of V234I, E254A, Y325Q, P334S, I427M, and Q473K, which involves changing the residues from the one found in the less stable 2B6dH to the more stable 2B1dH. 2B6dH mutants that show increased stability will be combined with L264F, and the resulting mutant with the highest expression and stability will be used as a template for directed evolution to model a thermostable 2B6dH. To investigate the functional role of the 2B6 variant, K262R, which shows altered activity with several drugs, the mutant was constructed in 2B6dH and L264F backgrounds. K262R and K262R/L264F were then characterized for stability and for functional properties using model and drug substrates/inhibitors. K262R showed decreased protein stability and altered substrate metabolism and inhibitor binding compared with 2B6dH. Structural analysis to understand how K262R alters enzyme functions is underway. [NIH grant ES03619 and Center grant ES06676]

Rational Engineering of Human Cytochrome P450 2B6 for Enhanced Expression and Stability: Importance of a Leu264→Phe Substitution

Despite the emerging importance of human P450 2B6 in xenobiotic metabolism, thorough biochemical and biophysical characterization has been impeded as a result of low expression in Escherichia coli. Comparison with similar N-terminal truncated and C-terminal His-tagged constructs (rat P450 2B1dH, rabbit 2B4dH, and dog 2B11dH) revealed that P450 2B6dH showed the lowest thermal stability, catalytic tolerance to temperature, and chemical stability against guanidinium chloride-induced denaturation. Eleven P450 2B6dH mutants were rationally engineered based on sequence comparison with the three other P450 2B enzymes and the solvent accessibility of residues in the ligand-free crystal structure of P450 2B4dH. L198M, L264F, and L390P showed approximately 3-fold higher expression than P450 2B6dH. L264F alone showed enhanced stability against thermal and chemical denaturation compared with P450 2B6dH and was characterized further functionally. L264F showed similar preferential inhibition by pyridine over imidazole derivatives as P450 2B6dH. The Leu(264)-->Phe substitution did not alter the K(s) for inhibitors or the substrate benzphetamine, the K(m) for 7-ethoxy-4-(trifluoromethyl)coumarin, or the benzphetamine metabolite profiles. The enhanced stability and monodisperse nature of L264F made it suitable for isothermal titration calorimetry studies. Interaction of 1-benzylimidazole with L264F yielded a clear binding isotherm with a distinctly different thermodynamic signature from P450 2B4dH. The inhibitor docked differently in the binding pocket of a P450 2B6 homology model than in 2B4, highlighting the different chemistry of the active site of these two enzymes. Thus, L264F is a good candidate to further explore the unique structure-function relationships of P450 2B6 using X-ray crystallography and solution thermodynamics.

Cytochromes P450 catalyze oxidation of α,β-unsaturated aldehydes

We sought to establish whether heme-thiolate monooxygenases oxidize, α,β-unsaturated aldehydes generated during lipid peroxidation. Several recombinant P450s co-expressed with NADPH:P450 oxidoreductase were surveyed for aldehyde oxidation activity with anthracene-9-carboxaldehyde and 4-hydroxy-trans-2-nonenal (HNE). Murine P4502c29, human P4503A4, human P4502B6, and rabbit P4502B4 were good catalysts of aldehyde oxidation to carboxylic acids. Other P450s (e.g., P4501A2, 2E1, and 2J2) did not oxidize these aldehydes. P4502c29 and P4503A4 displayed Km/S0.5 values of approx. 1–20μM. The product measured by HPLC that co-migrates with authentic 4-hydroxynonenoic acid (HNA) had a mass spectrum identical to the standard. Using P4502c29, HNE was a mixed-competitive inhibitor of anthracene-9-carboxaldehyde oxidation, suggesting that both aldehydes are substrates for P4502c29. Specific inhibitors of aldehyde dehydrogenases and P450 were used to assess their role in the metabolism of HNE in primary rat hepatocytes. Inhibitors of aldehyde dehydrogenase (cyanamide) inhibited HNA formation by 60% and together cyanamide and miconazole (P450) caused over 85% inhibition of HNA formation. P450s are significant participants in metabolism of endogenous and exogenous unsaturated aldehydes in primary rat hepatocytes.