The role of Ile87 of CYP158A2 in oxidative coupling reaction

Both CYP158A1 and CYP158A2 are able to catalyze an oxidative C-C coupling reaction producing biflaviolin or triflaviolin in Streptomyces coelicolor A3(2). The substrate-bound crystal structures of CYP158A2 and CYP158A1 reveal that the side chain of Ile87 in CYP158A2 points to the active site contacting the distal flaviolin molecule, however, the bulkier side chain of Lys90 in CYP158A1 (corresponding to Ile87 in CYP158A2) is toward the distal surface of the protein. These results suggest that these residues could be important in determining product regiospecificity. In order to explore the role of the two residues in catalysis, the reciprocal mutants, Ile87Lys and Lys90Ile, of CYP158A2 and CYP158A1, respectively, were generated and characterized. The mutant Ile87Lys enzyme forms two isomers of biflaviolin instead of three isomers of biflaviolin in wild-type CYP158A2. CYP158A1 containing the substitution of lysine with isoleucine has the same catalytic activity compared with the wild-type CYP158A1. The crystal structure of Ile87Lys showed that the BC loop in the mutant is in a very different orientation compared with the BC loop in both CYP158A1/A2 structures. These results shed light on the mechanism of the oxidative coupling reaction catalyzed by cytochrome P450.     

Binding of two flaviolin substrate molecules, oxidative coupling, and crystal structure of Streptomyces coelicolor A3(2) cytochrome P450 158A2

Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.     

Role of active site water molecules and substrate hydroxyl groups in oxygen activation by cytochrome P450 158A2: a new mechanism of proton transfer

From the x-ray crystal structure of CYP158A2 (Zhao, B., Guengerich, F. P., Bellamine, A., Lamb, D. C., Izumikawa, M., Lei, L., Podust, L. M., Sundaramoorthy, M., Reddy, L. M., Kelly, S. L., Kalaitzis, J. A., Stec, D., Voehler, M., Falck, J. R., Moore, B. S., Shimada, T., and Waterman, M. R. (2005) J. Biol. Chem. 280, 11599-11607), one of 18 cytochrome P450 (CYP) genes in the actinomycete Streptomyces coelicolor, ordered active site water molecules (WAT505, WAT600, and WAT640), and hydroxyl groups of the substrate flaviolin were proposed to participate in proton transfer and oxygen cleavage in this monooxygenase. To probe their roles in catalysis, we have studied the crystal structures of a substrate analogue (2-hydroxy-1,4-naphthoquinone) complex with ferric CYP158A2 (2.15 A) and the flaviolin ferrous dioxygen-bound CYP158A2 complex (1.8 A). Catalytic activity toward 2-hydroxy-1,4-naphthoquinone was approximately 70-fold less than with flaviolin. In the ferrous dioxygen-bound flaviolin complex, the three water molecules in the ferric flaviolin complex still occupy the same positions and form hydrogen bonds to the distal dioxygen atom. These findings suggest that CYP158A2 utilizes substrate hydroxyl groups to stabilize active site water and further assist in the iron-linked dioxygen activation. A continuous hydrogen-bonded water network connecting the active site to the protein surface (bulk solvent) not present in the other two ferrous dioxygen-bound P450 structures (CYP101A1/P450cam and CYP107A1/P450eryF) is proposed to participate in the proton-delivery cascade, leading to dioxygen bond scission. This ferrous-dioxygen structure suggests two classes of P450s based on the pathway of proton transfer, one using the highly conserved threonine in the I-helix (CYP101A1) and the other requiring hydroxyl groups of the substrate molecules either directly transferring protons (CYP107A1) or stabilizing a water pathway for proton transfer (CYP158A2).     

Ligand-assisted inhibition in cytochrome P450 158A2 from Streptomyces coelicolor A3(2)

Cytochrome P450 158A2 (CYP158A2) can polymerize flaviolin to red-brown pigments, which may afford physical protection to the organism, possibly against the deleterious effects of UV radiation. We have found that the small molecule malonic acid enables cocrystallization of this mixed function oxidase with the azole inhibitor 4-phenylimidazole. The presence of malonate molecules affects the behavior of the binding of 4-phenylimidazole to CYP158A2 and increases inhibition potency up to 2-fold compared to 4-phenylimidazole alone. We report here the crystal structure of the 4-phenylimidazole/malonate complex of CYP158A2 at 1.5 A. Two molecules of malonate used in crystallization are found above the single inhibitor molecule in the active site. Those two molecules are linked between the BC loop and beta 1-4/beta 6-1 strands via hydrogen bond interactions to stabilize the conformational changes of the BC loop and beta strands that take place upon inhibitor binding compared to the ligand-free structure we have reported previously. 4-Phenylimidazole can launch an extensive hydrogen-bonding network in the region of the F/G helices which may stabilize the conformational changes. Our findings clearly show that two molecules of malonate assist the inhibitor 4-phenylimidazole to assume a specific location producing more inhibition in the enzyme catalytic activity.     

cDNA cloning and characterization of feline CYP1A1 and CYP1A2

Deficiency of drug glucuronidation in the cat is one of the major reasons why this animal is highly sensitive to the side effects of drugs. The characterization of cytochrome P450 isoforms belonging to the CYP1A subfamily, which exhibit important drug oxidation activities such as activation of pro-carcinogens, was investigated. Two cDNAs, designated CYP1A-a and CYP1A-b, corresponding to the CYP1A subfamily were obtained from feline liver. CYP1A-a and CYP1A-b cDNAs comprise coding regions of 1554 bp and 1539 bp, and encode predicted amino acid sequences of 517 and 512 residues, respectively. These amino acid sequences contain a heme-binding cysteine and a conserved threonine. The cDNA identities, as well as the predicted amino acid sequences containing six substrate recognition sites, suggest that CYP1A-a and CYP1A-b correspond to CYP1A1 and CYP1A2, respectively. This was confirmed by the kinetic parameters of the arylhydrocarbon hydroxylase and 7-ethoxyresorufin O-deethylase activities of expressed CYPs in yeast AH22 cells and by the tissue distribution of each mRNA. However, theophylline 3-demethylation is believed to be catalyzed by CYP1A1 in cats, based on the high V(max) and low K(m) seen, in contrast to other animals. Because feline CYP1A2 had a higher K(m) for phenacetin O-deethylase activity with acetaminophen, which cannot be conjugated with glucuronic acid due to UDP-glucuronosyltransferase deficiency, it is supposed that the side effects of phenacetin as a result of toxic intermediates are severe and prolonged in cats.     

Expression, purification, and characterization of Bacillus subtilis cytochromes P450 CYP102A2 and CYP102A3: flavocytochrome homologues of P450 BM3 from Bacillus megaterium

The cyp102A2 and cyp102A3 genes encoding the two Bacillus subtilis homologues (CYP102A2 and CYP102A3) of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium have been cloned, expressed in Escherichia coli, purified, and characterized spectroscopically and enzymologically. Both enzymes contain heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors and bind a variety of fatty acid molecules, as demonstrated by conversion of the low-spin resting form of the heme iron to the high-spin form induced by substrate-binding. CYP102A2 and CYP102A3 catalyze the fatty acid-dependent oxidation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and reduction of artificial electron acceptors at high rates. Binding of carbon monoxide to the reduced forms of both enzymes results in the shift of the heme Soret band to 450 nm, confirming the P450 nature of the enzymes. Reverse-phase high-performance liquid chromatography (HPLC) of products from the reaction of the enzymes with myristic acid demonstrates that both catalyze the subterminal hydroxylation of this substrate, though with different regioselectivity and catalytic rate. Both P450s 102A2 and 102A3 show kinetic and binding preferences for long-chain unsaturated and branched-chain fatty acids over saturated fatty acids, indicating that the former two molecule types may be the true substrates. P450s 102A2 and 102A3 exhibit differing substrate selectivity profiles from each other and from P450 BM3, indicating that they may fulfill subtly different cellular roles. Titration curves for binding and turnover kinetics of several fatty acid substrates with P450s 102A2 and 102A3 are better described by sigmoidal (rather than hyperbolic) functions, suggesting binding of more than one molecule of substrate to the P450s, or possibly cooperativity in substrate binding. Comparison of the amino acid sequences of the three flavocytochromes shows that several important amino acids in P450 BM3 are not conserved in the B. subtilis homologues, pointing to differences in the binding modes for the substrates that may explain the unusual sigmoidal kinetic and titration properties.     

Tomato cytochrome P450 CYP734A7 functions in brassinosteroid catabolism

Several cytochrome P450 monooxygenases (P450s) catalyze essential oxidative reactions in brassinosteroid (BR) biosynthesis as well as in BR catabolism; however, only limited information exists on the P450s involved in the BR catabolic pathway. Here, we report the characterization of two P450 mRNAs, CYP734A7 and CYP734A8, from Lycopersicon esculentum. These P450s show high homology with Arabidopsis CYP734A1/BAS1 (formerly CYP72B1), which inactivates BRs via C-26 hydroxylation. Transgenic tobacco plants that constitutively overexpressed CYP734A7 showed an extreme dwarf phenotype similar to BR deficiency. Quantitative gas chromatography-mass spectrometry analysis of endogenous BRs in the transgenic plants showed that the levels of castasterone and 6-deoxocastasterone significantly decreased in comparison with those in wild-type plants. By measuring the Type I substrate-binding spectra using recombinant CYP734A7, the dissociation constants for castasterone, brassinolide, and 6-deoxocastasterone were determined to be 6.7, 12, and 12 microM, respectively. In an in vitro assay, CYP734A7 was confirmed to metabolize castasterone to 26-hydroxycastasterone. In addition, 28-norcastasterone and brassinolide were converted to the hydroxylated products. The expression of CYP734A7 and CYP734A8 genes in tomato seedlings was upregulated by exogenous application of bioactive BRs. These results indicated that CYP734A7 is a C-26 hydroxylase of BRs and is likely involved in BR catabolism in tomato. The presence of the CYP734A subfamily in various plant species suggests that oxidative inactivation of BRs by these proteins is a widespread phenomenon in plants.     

Synthesis of halogenated pregnanes, mechanistic probes of steroid hydroxylases CYP17A1 and CYP21A2

The human steroidogenic cytochromes P450 CYP17A1 (P450c17, 17α-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) are required for the biosynthesis of androgens, glucocorticoids, and mineralocorticoids. Both enzymes hydroxylate progesterone at adjacent, distal carbon atoms and show limited tolerance for substrate modification. Halogenated substrate analogs have been employed for many years to probe cytochrome P450 catalysis and to block sites of reactivity, particularly for potential drugs. Consequently, we developed efficient synthetic approaches to introducing one or more halogen atom to the 17- and 21-positions of progesterone and pregnenolone. In particular, novel 21,21,21-tribromoprogesterone and 21,21,21-trichloroprogesterone were synthesized using the nucleophilic addition of either bromoform or chloroform anion onto an aldehyde precursor as the key step to introduce the trihalomethyl moieties. When incubated with microsomes from yeast expressing human CYP21A2 or CYP17A1 with P450-oxidoreductase, CYP21A2 metabolized 17-fluoroprogesterone to a single product, whereas incubations with CYP17A1 gave no products. Halogenated steroids provide a robust system for exploring the substrate tolerance and catalytic plasticity of human steroid hydroxylases.     

Cloning,expression and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Cytochrome P450 monooxygenases of the CYP102A subfamily are single-component natural fusion proteins consisting of a heme domain and a diflavin reductase. The characterised CYP102A enzymes are fatty acid hydroxylases with turnover rates of several thousands per minute. In search of new P450s with similar activities, but with a broader substrate spectrum, we cloned, expressed and characterised CYP102A7 from Bacillus licheniformis. As expected, CYP102A7 was active towards medium-chain fatty acids but showed a strong preference for saturated over unsaturated fatty acids, which could not be observed for either of the CYP102A members so far. Besides fatty acids, CYP102A7 was able to catalyse the oxidation of cyclic and acyclic terpenes with high activity and coupling efficiency. For example, (R)-(+)-limonene was converted with activity of 220 nmol nmol P450(-1) min(-1) and 80% coupling. Unusual for enzymes of the CYP102A subfamily was the deethylation activity of CYP102A7 towards 7-ethoxycoumarin. Furthermore, this monooxygenase, though having a moderate thermal stability, exhibited 50% of its initial activity in the presence of 26% DMSO. Comparison of the homology models of CYP102A7 and other members of the CYP102A subfamily revealed distinct differences in the shape of the substrate access channel and the active site, which might explain differences in catalytic properties of these homologous enzymes.     

Characterization of differences in substrate specificity among CYP1A1, CYP1A2 and CYP1B1: an integrated approach employing molecular docking and molecular dynamics simulations

Recent trends in new drug discovery of anticancer drugs have made oncologists more aware of the fact that the new drug discovery must target the developing mechanism of tumorigenesis to improve the therapeutic efficacy of antineoplastic drugs. The drugs designed are expected to have high affinity towards the novel targets selectively. Current research highlights overexpression of CYP450s, particularly cytochrome P450 1A1 (CYP1A1), in tumour cells, representing a novel target for anticancer therapy. However, the CYP1 family is identified as posing significant problems in selectivity of anticancer molecules towards CYP1A1. Three members have been identified in the human CYP1 family: CYP1A1, CYP1A2 and CYP1B1. Although sequences of the three isoform have high sequence identity, they have distinct substrate specificities. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics, design novel antitumour compounds that could be specifically metabolized by only CYP1A1 to mediate their antitumour activity and elucidate the reasons for differences in substrate specificity profile among the three proteins. In the present study, we employed a combination of computational methodologies: molecular docking and molecular dynamics simulations. We utilized eight substrates for elucidating the difference in substrate specificity of the three isoforms. Lastly, we conclude that the substrate specificity of a particular substrate depends upon the type of the active site residues, the dynamic motions in the protein structure upon ligand binding and the physico‐chemical characteristics of a particular ligand. Copyright © 2016 John Wiley & Sons, Ltd.     

Molecular cloning and characterization of CYP80G2, a cytochrome P450 that catalyzes an intramolecular C-C phenol coupling of (S)-reticuline in magnoflorine biosynthesis, from cultured Coptis japonica cells

Cytochrome P450s (P450) play a key role in oxidative reactions in plant secondary metabolism. Some of them, which catalyze unique reactions other than the standard hydroxylation, increase the structural diversity of plant secondary metabolites. In isoquinoline alkaloid biosyntheses, several unique P450 reactions have been reported, such as methylenedioxy bridge formation, intramolecular C-C phenol-coupling and intermolecular C-O phenol-coupling reactions. We report here the isolation and characterization of a C-C phenol-coupling P450 cDNA (CYP80G2) from an expressed sequence tag library of cultured Coptis japonica cells. Structural analysis showed that CYP80G2 had high amino acid sequence similarity to Berberis stolonifera CYP80A1, an intermolecular C-O phenol-coupling P450 involved in berbamunine biosynthesis. Heterologous expression in yeast indicated that CYP80G2 had intramolecular C-C phenol-coupling activity to produce (S)-corytuberine (aporphine-type) from (S)-reticuline (benzylisoquinoline type). Despite this intriguing reaction, recombinant CYP80G2 showed typical P450 properties: its C-C phenol-coupling reaction required NADPH and oxygen and was inhibited by a typical P450 inhibitor. Based on a detailed substrate-specificity analysis, this unique reaction mechanism and substrate recognition were discussed. CYP80G2 may be involved in magnoflorine biosynthesis in C. japonica, based on the fact that recombinant C. japonica S-adenosyl-L-methionine:coclaurine N-methyltransferase could convert (S)-corytuberine to magnoflorine.     

Metabolism of 4-n-nonylphenol by non-modified and CYP1A1- and CYP1A2-transgenic cell cultures of tobacco

The metabolism of 14C-4-n-nonylphenol (l4C-4-n-NP), as a model for the xenoestrogen nonylphenol, was investigated in three types of tobacco cell suspension cultures: one genetically non-modified culture (NT) and two cultures constitutively expressing human cytochrome P450 CYP1A1 or CYP1A2. With 1 mg l(-1) of 14C-4-n-NP and 24 h of incubation, the xenobiotic was transformed almost completely to glycosides. After glycosidic cleavage, 14C-4-n-NP and several primary metabolites of 4C-4-n-NP were liberated. Portions of the primary metabolites were 29.3% (NT culture), 34.3% (CYP1A1 culture), and 50.7% of applied 14C (CYP1A2 culture). Thus, the endogenous capacity of the tobacco cells to form primary metabolites of 4-n-NP was noticeably higher than that of CYP1A1 or CYP1A2. The results however clearly suggest that 4-n-NP is - even though a poor - substrate of CYP1A1 and CYP1A2. In order to examine metabolic profiles of 4-n-NP in the NT, CYP1A1 and CYP1A2 cultures, the suspensions were exposed to 10 mg 1(-1) of 14C-4-n-NP using a two-liquid-phase system with carrier n-hexadecane and 192 h of incubation. Results obtained resembled those of the low concentration study. The oxidative metabolic profiles determined after hydrolytic cleavage using GC-EIMS were similar in the NT, CYP1A1 and CYP1A2 cultures. Main metabolites were side-chain mono-hydroxylated derivatives of 4-n-NP with 6'-, 7'- and 8'-OH-4-n-NP as prominent metabolites. In addition, olefinic side-chain hydroxy, ring methoxylated, keto and ring hydroxylated derivatives were observed. The lack of differences in metabolic profiles among the CYP1A1, CYP1A2 and NT cultures was referred to the low enzymatic activity of CYP1A1 and CYP1A2 as compared to the higher endogenous oxidative capacity of tobacco, as well as to similar metabolic profiles of 4-n-NP produced by CYP1A1 and CYP1A2 and tobacco itself.     

Metabolic pathways of dioxin by CYP1A1: species difference between rat and human CYP1A subfamily in the metabolism of dioxins

Metabolism of polychlorinated dibenzo-p-dioxins by CYP1A subfamily was examined by using the recombinant yeast microsomes. In substrate specificity and reaction specificity, considerable species differences between rats and humans were observed in both CYP1A1- and CYP1A2-dependent metabolism of dioxins. Among four CYPs, rat CYP1A1 showed the highest activity toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. To reveal the mechanism of dioxin metabolism, we examined rat CYP1A1-dependent metabolism of 2-chloro-dibenzo-p-dioxin. In addition to hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and cleavage of an ether linkage of the dioxin ring were observed. In particular, the cleavage of an ether linkage of the dioxin ring appeared most important for the detoxication of dioxins. Based on these results, the metabolic pathways of 2-chloro-dibenzo-p-dioxin by rat CYP1A1 were proposed. The metabolic pathways contain most of the metabolites observed in vivo using experimental animals, suggesting that P450 monooxygenase systems including CYP1A1 are greatly responsible for dioxin metabolism in vivo.     

A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of all-trans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA.     

Heteroactivator effects on the coupling and spin state equilibrium of CYP2C9

The cytochromes P450 are capable of oxidizing a variety of xenobiotics. Binding of a small molecule heteroactivator to a P450 can alter the coupling of substrate oxidation during P450 catalysis, but the degree to which coupling or shunting via one of the three catalytic cycle branch points is linked to the heteroactivator-modified position of bound substrate is unknown. Using reconstituted CYP2C9, stoichiometric measurements were gathered with three substrates and two classes of heteroactivators to further understand the mechanisms involved in heteroactivation. Heteroactivation of P450 metabolism appeared to involve, but not require, changes in coupling and that increased uncoupling to a specific byproduct like H(2)O(2) does not necessarily correlate to the degree of coupling. In addition, spectroscopy demonstrated that every heteroactivator tested influenced the spin equilibrium of the heme iron even in the presence of saturating substrate suggesting that both substrate proximity and the ability to desolvate the heme can be involved in heteroactivation.     

Cloning and characterization of a novel CYP3A1 allelic variant: analysis of CYP3A1 and CYP3A2 sex-hormone-dependent expression reveals that the CYP3A2 gene is regulated by testosterone.

A clone was isolated from a cDNA library constructed from phenobarbital-treated Wistar rat liver and proven to correspond to the full-length mRNA of a polymorphic variant of Sprague-Dawley CYP3A1. Eight nucleotide differences were detected in a single 76-nucleotide stretch and confirmed to be present in the genomic clone. They are seated in a region implicated in the definition of a substrate binding domain of the native P450. Three out of the eight nucleotide changes are nonconservative, implicating the replacement of Thr/Ala 207, Phe/Ile 213, and Ile/Val 232. This is the first report of an allelic variant of CYP3A1, a new example of interstrain P450 variability. The CYP3A subfamily is composed of several genes coding for active testosterone 6 beta-hydroxylases which are expressed in the liver. CYP3A genes are under strong and distinct developmental regulation. Conversely to CYP3A1, transiently expressed in immature animals, CYP3A2 is constitutively expressed in the liver early after birth and characterized by an extinction in the adult females. Castration of 90-day-old male rats causes a drastic reduction (80%) of CYP3A2 mRNA relative abundance. Administration of testosterone propionate restores the physiological levels of CYP3A2 mRNA characteristic of the male rat liver. Our results demonstrate the existence of a direct relationship between the male hormonal status and the constitutive expression of rat liver CYP3A2.     

Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation

Hepatoma cells show alterations in the response to oxidative stress (decreased lipid peroxidation) and in xenobiotic metabolism enzymes (decreased P450, increased GST and ALDH3). This study examined the effect of lipid peroxidation on the expression of the above enzymes in two rat hepatoma cell lines (MH(1)C(1) and 7777). To induce oxidative stress, cells were exposed to arachidonic acid (to increase lipid peroxidation substrate) and/or to beta-naphthoflavone (to increase CYP450), and treated with one dose of iron/histidine. The cells, that were still viable after the challenge, were refed with the culture medium and CYP1A1, GST, and ALDH3 enzymes monitored for 1, 6, 12, and 24 h. Treatments that increased markers indicative of lipid peroxidation are associated with a decrease in enzyme activities, which was permanent for CYP1A1 and transient for the other enzymes. We speculate from these data that aldehydic byproducts of lipid peroxidation may be responsible for these effects. Thus, restoration of lipid peroxidation in hepatoma cells seems to induce a rapid adaptation to oxidative stress, which is achieved by a simultaneous decrease of reactive oxygen species production and an increase in the two main enzymes involved in the removal of the aldehydic products of lipid peroxidation.     

Role of residue 87 in substrate selectivity and regioselectivity of drug-metabolizing cytochrome P450 CYP102A1 M11

CYP102A1, originating from Bacillus megaterium, is a highly active enzyme which has attracted much attention because of its potential applicability as a biocatalyst for oxidative reactions. Previously we developed drug-metabolizing mutant CYP102A1 M11 by a combination of site-directed and random mutagenesis. CYP102A1 M11 contains eight mutations, when compared with wild-type CYP102A1, and is able to produce human-relevant metabolites of several pharmaceuticals. In this study, active-site residue 87 of drug-metabolizing mutant CYP102A1 M11 was mutated to all possible natural amino acids to investigate its role in substrate selectivity and regioselectivity. With alkoxyresorufins as substrates, large differences in substrate selectivities and coupling efficiencies were found, dependent on the nature of residue 87. For all combinations of alkoxyresorufins and mutants, extremely fast rates of NADPH oxidation were observed (up to 6,000 min⁻¹). However, the coupling efficiencies were extremely low: even for the substrates showing the highest rates of O-dealkylation, coupling efficiencies were lower than 1%. With testosterone as the substrate, all mutants were able to produce three hydroxytestosterone metabolites, although with different activities and with remarkably different product ratios. The results show that the nature of the amino acid at position 87 has a strong effect on activity and regioselectivity in the drug-metabolizing mutant CYP102A1 M11. Because of the wide substrate selectivity of CYP102A1 M11 when compared with wild-type CYP102A1, this panel of mutants will be useful both as biocatalysts for metabolite production and as model proteins for mechanistic studies on the function of P450s in general.     

Effects of ionic strength on the functional interactions between CYP2B4 and CYP1A2

The presence of one P450 can influence the catalytic characteristics of a second enzyme through the formation of heteromeric P450 complexes. Such a complex has been reported for mixed reconstituted systems containing NADPH-cytochrome P450 reductase, CYP2B4, and CYP1A2, where a dramatic inhibition of 7-pentoxyresorufin-O-dealkylation (PROD) was observed when compared to simple reconstituted systems containing reductase and a single P450 enzyme. The goal of the present study was to characterize this interaction by examining the potential of the CYP1A2-CYP2B4 complex to be formed by charge-pair interactions. With ionic interactions being sensitive to the surrounding ionic environment, monooxygenase activities were measured in both simple systems and mixed reconstituted systems as a function of ionic strength. PROD was found to be decreased at high ionic strength in both simple and mixed reconstituted systems, due to disruption of reductase-P450 complexes. Additionally, the inhibition of PROD in mixed reconstituted systems was relieved at high ionic strength, consistent with disruption of the CYP2B4-CYP1A2 complex. When ionic strength was measured as a function of CYP1A2 concentration, a shift to the right in the inflection point of the biphasic curve occurred at high ionic strength, consistent with a loss in CYP1A2 affinity for CYP2B4. When this analysis was applied to the same systems using a different substrate, 7-EFC, evidence for a high-affinity complex was not observed, demonstrating that the characteristics of the CYP1A2-CYP2B4 complex are influenced by the substrates present. These results support the role for a substrate specific electrostatic interaction between these P450 enzymes.