Raku Shinkyo

Molecular Cloning and Characterization of CYP719, a Methylenedioxy Bridge-forming Enzyme That Belongs to a Novel P450 Family, from cultured Coptis japonica Cells

Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3′-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3′-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.

Conversion of 7-Dehydrocholesterol to 7-Ketocholesterol Is Catalyzed by Human Cytochrome P450 7A1 and Occurs by Direct Oxidation without an Epoxide Intermediate

7-Ketocholesterol is a bioactive sterol, a potent competitive inhibitor of cytochrome P450 7A1, and toxic in liver cells. Multiple origins of this compound have been identified, with cholesterol being the presumed precursor. Although routes for formation of the 7-keto compound from cholesterol have been established, we found that 7-dehydrocholesterol (the immediate precursor of cholesterol) is oxidized by P450 7A1 to 7-ketocholesterol (kcat/Km = 3 × 104 m−1 s−1). P450 7A1 converted lathosterol (Δ5-dihydro-7-dehydrocholesterol) to a mixture of the 7-keto and 7α,8α-epoxide products (∼1:2 ratio), with the epoxide not rearranging to the ketone. The oxidation of 7-dehydrocholesterol occured with predominant formation of 7-ketocholesterol and with the 7α,8α-epoxide as only a minor product; the synthesized epoxide was stable in the presence of P450 7A1. The mechanism of 7-dehydrocholesterol oxidation to 7-ketocholesterol is proposed to involve a FeIII-O-C-C+ intermediate and a 7,8-hydride shift or an alternative closing to yield the epoxide (Liebler, D. C., and Guengerich, F. P. (1983) Biochemistry 22, 5482–5489). Accordingly, reaction of P450 7A1 with 7-[2H1]dehydrocholesterol yielded complete migration of deuterium in the product 7-ketocholesterol. The finding that 7-dehydrocholesterol is a precursor of 7-ketocholesterol has relevance to an inborn error of metabolism known as Smith-Lemli-Opitz syndrome (SLOS) caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase, the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol in tissues and fluids of SLOS patients. Our findings suggest that 7-ketocholesterol levels may also be elevated in SLOS tissue and fluids as a result of P450 7A1 oxidation of 7-dehydrocholesterol.

Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily.

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by recombinant yeast cells expressing either rat CYP1A1 or CYP1A2 was examined. When each of the dibenzo-p-dioxins (DDs), mono-, di-, and tri-chloroDDs, was added to the cell culture of the recombinant yeast, a remarkable metabolism was observed. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, hydroxylation with elimination of a chloride substituent, and opening of dioxin ring. The distinct difference was observed in substrate specificity and reaction specificity between CYP1A1 and CYP1A2. Kinetic analysis using microsomal fractions prepared from the recombinant yeast cells revealed that 2,7-dichloroDD and 2,3,7-trichloroDD were good substrates for both CYP1A1 and CYP1A2. When 2,3,7-trichloroDD was added to the yeast cells expressing each of rat CYP1A1 and CYP1A2, most of 2,3,7-trichloroDD was first converted to 8-hydroxy-2,3,7-trichloroDD, and further metabolized to more hydrophilic compounds whose ethereal bridges were cleaved. These findings give essential information on the metabolism of PCDDs in mammalian liver. In addition, this study indicates the possibility of application of microorganisms expressing mammalian cytochrome P450 to bioremediation of contaminated soils with dioxins.

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.

Cytochrome P450 7A1 Cholesterol 7α-Hydroxylation INDIVIDUAL REACTION STEPS IN THE CATALYTIC CYCLE AND RATE-LIMITING FERRIC IRON REDUCTION

Cytochrome P450 (P450) 7A1 is well known as the cholesterol 7α-hydroxylase, the first enzyme involved in bile acid synthesis from cholesterol. The human enzyme has been reported to have the highest catalytic activity of any mammalian P450. Analyses of individual steps of cholesterol 7α-hydroxylation reaction revealed several characteristics of this reaction: (i) two-step binding of cholesterol to ferric P450, with an apparent Kd of 0.51 μm, (ii) a rapid reduction rate in the presence of cholesterol (∼10 s−1 for the fast phase), (iii) rapid formation of a ferrous P450-cholesterol-O2 complex (29 s−1), (iv) the lack of a non-competitive kinetic deuterium isotope effect, (v) the lack of a kinetic burst, and (vi) the lack of a deuterium isotope effect when the reaction was initiated with the ferrous P450-cholesterol complex. A minimum kinetic model was developed and is consistent with all of the observed phenomena and the rates of cholesterol 7α-hydroxylation and H2O and H2O2 formation. The results indicate that the first electron transfer step, although rapid, becomes rate-limiting in the overall P450 7A1 reaction. This is a different phenomenon compared with other P450s that have much lower rates of catalysis, attributed to the much more efficient substrate oxidation steps in this reaction.Cytochrome P450 (P450) 7A1 is well known as the cholesterol 7α-hydroxylase, the first enzyme involved in bile acid synthesis from cholesterol. The human enzyme has been reported to have the highest catalytic activity of any mammalian P450. Analyses of individual steps of cholesterol 7α-hydroxylation reaction revealed several characteristics of this reaction: (i) two-step binding of cholesterol to ferric P450, with an apparent K(d) of 0.51 μM, (ii) a rapid reduction rate in the presence of cholesterol (∼10 s(-1) for the fast phase), (iii) rapid formation of a ferrous P450-cholesterol-O(2) complex (29 s(-1)), (iv) the lack of a non-competitive kinetic deuterium isotope effect, (v) the lack of a kinetic burst, and (vi) the lack of a deuterium isotope effect when the reaction was initiated with the ferrous P450-cholesterol complex. A minimum kinetic model was developed and is consistent with all of the observed phenomena and the rates of cholesterol 7α-hydroxylation and H(2)O and H(2)O(2) formation. The results indicate that the first electron transfer step, although rapid, becomes rate-limiting in the overall P450 7A1 reaction. This is a different phenomenon compared with other P450s that have much lower rates of catalysis, attributed to the much more efficient substrate oxidation steps in this reaction.

Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 and its mutant

The metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 BM-3 from Bacillus megaterium and a mutant enzyme of it (AL4V; Ala74Gly, Phe87Val, Leu188Gln triple mutant) was examined. Both purified enzymes metabolized 1-monochloro-, 2,3-dichloro-, and 2,3,7-trichloro-dibenzo-p-dioxin, but not 2,3,7,8-tetrachloro-dibenzo-p-dioxin. The mutant AL 4V had 2–12 times higher activity than the wild-type P450 BM-3 towards polychlorinated dibenzo-p-dioxins. The products were hydroxylated at an unsubstituted position and/or showing migration of the chloride and were less toxic derivatives with lower than 10% toxicity of the original compounds.

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.

Cytochrome P450 2S1 is Reduced by NADPH-Cytochrome P450 Reductase

Cytochrome P450 (P450) 2S1 is one of the orphan P450s without a clear physiological function. Controversy has arisen as to whether it can interact with NADPH-P450 reductase and accept electrons. The reduction of 1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (AQ4N) by P450 2S1 was confirmed, and the NADPH consumption rates were measured aerobically and anaerobically in the absence and presence of the drug. The reduction kinetics of P450 2S1 were rapid, as measured by stopped-flow kinetics. These results confirm that P450 2S1 can be reduced by NADPH-P450 reductase and suggest normal mixed-function oxidase roles of P450 2S1 to be revealed.

Inhibition of Human Cytochrome P450 3A4 by Cholesterol

Cholesterol has been shown to be hydroxylated at the 4β-position by cytochrome P450 3A4, and the reaction occurs in vivo (Bodin, K., Andersson, U., Rystedt, E., Ellis, E., Norlin, M., Pikuleva, I., Eggertsen, G., Björkhem, I., and Diczfalusy, U. (2002) J. Biol. Chem. 277, 31534–31540). If cholesterol is a substrate of P450 3A4, then it follows that it should also be an inhibitor, particularly in light of the high concentrations found in liver. Heme perturbation spectra indicated a Kd value of 8 μm for the P450 3A4-cholesterol complex. Cholesterol inhibited the P450 3A4-catalyzed oxidations of nifedipine and quinidine, two prototypic substrates, in liver microsomes and a reconstituted enzyme system with Ki ∼ 10 μm in an apparently non-competitive manner. The concentration of cholesterol could be elevated 4–6-fold in cultured human hepatocytes by incubation with cholesterol; the level of P450 3A4 and cell viability were not altered under the conditions used. Nifedipine oxidation was inhibited when the cholesterol level was increased. We conclude that cholesterol is both a substrate and an inhibitor of P450 3A4, and a model is presented to explain the kinetic behavior. We propose that the endogenous cholesterol in hepatocytes should be considered in models of prediction of metabolism of drugs and steroids, even in the absence of changes in the concentrations of free cholesterol.

Generation of 2,3,7,8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis☆

Polychlorinated dibenzo-p-dioxins (PCDDs) are known as g environmental contaminants on account of the extreme toxicity. Among these compounds, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is regarded as the most toxic one. The extremely high toxicity of 2,3,7,8-TetraCDD is based on its high affinity for Ah receptor and nearly undetectable metabolism in mammalian body. Based on our previous studies, we assumed that enlarging the space of substrate-binding pocket of rat CYP1A1 might generate the catalytic activity toward 2,3,7,8-TetraCDD. Large-sized amino acid residues located at putative substrate-binding sites of rat CYP1A1 were substituted for alanine by site-directed mutagenesis. Among eight mutants examined, the mutant in the putative F-G loop, F240A, showed metabolic activity toward 2,3,7,8-TetraCDD. HPLC and GC-MS analyses strongly suggested that the metabolite was 8-hydroxy-2,3,7-TriCDD. Ah receptor assay revealed that the affinity of 8-hydroxy-2,3,7-TriCDD for Ah receptor was less than 0.01% of 2,3,7,8-TetraCDD, indicating that the F240A-dependent metabolism resulted in remarkable detoxification of 2,3,7,8-TetraCDD. The novel 2,3,7,8-TetraCDD-metabolizing enzyme could be applicable to bioremediation of contaminated soils with dioxin, elimination of dioxin from foods, and clinical treatment for people who accidentally take dioxin into their systems.