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CYP105A1 from Streptomyces griseolus converts vitamin D3 to its biologically active form, 1α,25-dihydroxy vitamin D3. R73A/R84A mutation enhanced the 1α- and 25-hydroxylation activity for vitamin D3, while M239A mutation generated the 1α-hydroxylation activity for vitamin D2. In this study, the stability of six CYP105A1 enzymes, including 5 variants (R73A/R84A, M239A, R73A/R84A/M239A (=TriA), TriA/E90A, and TriA/E90D), was examined. Circular dichroism analysis revealed that M239A markedly reduces the enzyme stability. Protein fluorescence analysis disclosed that these mutations, especially M239A, induce large changes in the local conformation around Trp residues. Strong stabilizing effect of glycerol was observed. Nondenaturing PAGE analysis showed that CYP105A1 enzymes are prone to self-association. Fluorescence analysis using a hydrophobic probe 8-anilino-1-naphthalenesulfonic acid suggested that M239A mutation enhances self-association and that E90A and E90D mutations, in cooperation with M239A, accelerate self-association with little effect on the stability., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Cytochrome P450 (CYP) enzymes constitute a superfamily of heme-containing monooxygenases. CYPs are involved in the metabolism of many chemicals such as drugs and agrochemicals. Therefore, examining the metabolic reactions by each CYP isoform is important to elucidate their substrate recognition mechanisms. The clarification of these mechanisms may be useful not only for the development of new drugs and agrochemicals, but also for risk assessment of chemicals. In our previous study, we identified the metabolites of tebufenozide, an insect growth regulator, formed by two human CYP isoforms: CYP3A4 and CYP2C19. The accessibility of each site of tebufenozide to the reaction center of CYP enzymes and the susceptibility of each hydrogen atom for metabolism by CYP enzymes were evaluated by a docking simulation and hydrogen atom abstraction energy estimation at the density functional theory level, respectively. In this study, the same in silico prediction method was applied to the metabolites of tebufenozide derivatives by major human CYPs (CYP1A2, 2C9, 2C19, 2D6, and 3A4). In addition, the production rate of the metabolites by CYP3A4 was quantitively analyzed by frequency based on docking simulation and hydrogen atom abstraction energy using the classical QSAR approach. Then, the obtained QSAR model was applied to predict the sites of metabolism and the metabolite production order by each CYP isoform., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Ltd.)

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D 3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Our previous studies revealed that CYP105A1 can convert vitamin D 3 (VD3) to its active form, 1α,25-dihydroxyvitamin D 3 (1,25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast, these variants had a low ability to convert vitamin D 2 (VD2) to 1α,25-dihydroxyvitamin D 2 (1,25D2), whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25,26D2. A comparison of the docking models of 25D2 and 25D3 into the substrate-binding pocket of R73A/R84A suggests that the side chain of the Met239 inhibits the binding of 25D2 for 1α-hydroxylation. Therefore, the Met239 residue of R73A/R84A was substituted for Ala. As expected, the triple variant R73A/R84A/M239A showed a 22-fold higher 1α-hydroxylation activity towards 25D2. To the best of our knowledge, this is the first report on the generation of microbial cytochrome P450 that converts VD2 to 1,25D2 via 25D2., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Polychlorinated biphenyls (PCBs) accumulate in mammals via the food chain because of their characteristics such as slow degradation and high hydrophobicity. One of the 209 PCB congeners, 2,3',4,4',5-pentachlorobiphenyl (CB118), is abundantly found in the environment and in mammals. Understanding the metabolic fate of CB118 can provide important information toward evaluating its toxicity. In vitro studies on the metabolism of CB118 by cytochrome P450 enzymes (P450 or CYP) revealed that human CYP2B6 and rat CYP2B1 primarily metabolized it to 3-hydroxy (OH)-CB118, whereas rat CYP1A1 metabolized CB118 to 4-hydroxy-2,3,3',4',5-pentachlorobiphenyl (4-OH-CB107). Docking models of CYP2Bs with CB118 revealed a short distance between the 3-position of CB118 and the heme iron caused by polarization of the substrate-binding cavity, and maintenance of this pose through interaction with the peripheral amino acids determines the activity and position of hydroxylation. 4-Hydroxylation by rat CYP1A1 occurs owing to the longitudinal shape of the substrate-binding cavity toward the heme of CYP1A1. The metabolites 3-OH-CB118 and 4-OH-CB107 decreased potential for activating the aryl hydrocarbon receptor compared with that of CB118, thereby leading to a decrease in dioxin-like toxicity; however, the neurodevelopmental toxicity of 4-OH-CB107 has been previously reported. The results suggest that these 3 P450 isoforms play an important role in determining the extent of CB118 toxicity. This study will contribute to understanding of the metabolic fates and toxicity of CB118 in vivo., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study, we found that both the double variants were also capable of converting vitamin D2 to its active form, that is, 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), via 25(OH)D2, whereas its 1α-hydroxylation activity toward 25(OH)D2 was much lower than that toward 25(OH)D3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)2D2 that is one of the metabolites of vitamin D2 detected in human serum., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Polychlorinated dibenzo-p-dioxins (PCDDs) and coplanar polychlorinated biphenyls (PCBs) contribute to dioxin toxicity in humans and wildlife after bioaccumulation through the food chain from the environment. The authors examined human and rat cytochrome P450 (CYP)-dependent metabolism of PCDDs and PCBs. A number of human CYP isoforms belonging to the CYP1 and CYP2 families showed remarkable activities toward low-chlorinated PCDDs. In particular, human CYP1A1, CYP1A2, and CYP1B1 showed high activities toward monoCDDs, diCDDs, and triCDDs but no detectable activity toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetraCDD). Large amino acids located at putative substrate-recognition sites and the F-G loop in rat CYP1A1 contributed to the successful metabolism of 2,3,7,8-tetraCDD. Rat, but not human, CYP1A1 metabolized 3,3',4,4',5-pentachlorobiphenyl (CB126) to two hydroxylated metabolites. These metabolites are probably less toxic than is CB126, due to their higher solubility. Homology models of human and rat CYP1A1s and CB126 docking studies indicated that two amino acid differences in the CB126-binding cavity were important for CB126 metabolism. In this review, the importance of CYPs in the metabolism of dioxins and PCBs in mammals and the species-based differences between humans and rats are described. In addition, the authors reveal the molecular mechanism behind the binding modes of dioxins and PCBs in the heme pocket of CYPs.

Cytochrome P450 (CYP) of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR) activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene.

Dioxins, including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans, and coplanar polychlorinated biphenyls, are known to be metabolized by enzymes such as cytochrome (CYP) P450, angular dioxygenase, lignin peroxidase, and dehalogenase. It is noted that all of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase each have a distinct electron transport chain. Among these enzyme systems, we have focused on cytochrome P450-dependent metabolism of dioxins from the viewpoint of practical use for bioremediation. Mammalian and fungal cytochromes P450 showed remarkable activity toward low-chlorinated PCDDs. In particular, mammalian cytochromes P450 belonging to the CYP1 family showed high activity. Rat CYP1A1 showed high activity toward 2,3,7-trichloro-dibenzo-p-dioxin but no detectable activity for 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD). On the basis of these results, we assumed that enlarging the space of the substrate-binding pocket of rat CYP1A1 might generate TCDD-metabolizing enzyme. Large-sized amino acids located at putative substrate-recognition sites and F-G loop were substituted for alanine by site-directed mutagenesis. Finally, we successfully generated 2,3,7,8-TCDD-metabolizing enzyme by site-directed mutagenesis of rat CYP1A1. We hope that recombinant microorganisms harboring genetically engineered cytochrome P450 will be used for bioremediation of soil contaminated with PCDDs, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls in the future., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Recent progress on the application of cytochrome P450 (P450) to bioconversion processes, biosensors, and bioremediation were reviewed. Because regio- and enantioselective hydroxylation makes chemical synthesis difficult, a bioconversion process using P450 would be quite attractive. One of the most successful industrial applications of P450 may be the bioconversion process for pravastatin formation using a Streptomyces carbophilus CYP105A3. Unfortunately, practical application of P450s in the bioconversion process is limited because of their low stability, low activity and co-factor dependency. However, directed evolution is expected to generate useful P450 biocatalysts for a wide range of substrates. Shunt pathways of CYP152A1, CYP152A2, and CYP152B1 are notably promising for practical application, because these P450s require neither reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) nor electron donor proteins, and efficiently catalyze using hydrogen peroxide. A P450 biosensor using biochip technology is expected to become a tool for rapidly determining drugs and endogenous substances in plasma at a low cost. Bioremediation of dioxins and polychlorinated biphenyls (PCBs) by the CYP1 family appears to be possible by using suicidal, genetically engineered microorganisms. The P450 superfamily has tremendous potential for practical applications in various fields.

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)(2)D], interacts with vitamin D receptor (VDR) and induces antiproliferative, anti-invasive, proapoptotic and pro-differentiation activities in prostate cancer cells. Three cytochrome P-450 (CYP) hydroxylases are responsible for vitamin D synthesis and degradation, including vitamin D-25-hydroxylase (25-OHase) in the liver, and 25(OH)D-1α-hydroxylase (1α-OHase) or CYP27B1, and 25(OH)D-24-hydroxylase (24-OHase) or CYP24A1 in the kidneys. However, it is now recognized that CYP27B1 and CYP24A1 are also expressed in many tissues and cells, including the prostate. Although at least six CYP enzymes have been identified with 25-OHase activity, the two major ones are CYP27A1 and CYP2R1, and both are expressed in the prostate, with CYP2R1 as the predominate type. This indicates that prostate tissue has the ability to activate and inactivate vitamin D in an autocrine/paracrine fashion. Recent evidence indicates that 25-hydroxyvitamin D [25(OH)D] and its analogs can bind to VDR as agonists, without converting them to 1α,25(OH)(2)D or the corresponding 1α-hydroxylated metabolites, to modulate gene expressions that will lead to cell growth arrest and other antitumor activities. This finding suggests that the circulating levels of 25(OH)D, and the autocrine synthesis of 25(OH)D may play an important role in regulating the growth of prostate cancer. Thus, in addition to 1α,25(OH)(2)D analogs, the presence of CYP2R1, CYP27B1 and CYP24A1 in the prostate suggests that the analogs of vitamin D and 25(OH)D, especially those that are resistant to CYP24A1 degradation, can be developed and used for the prevention and treatment of prostate cancer.

Our previous study revealed that CYP2C9 played a central role in sesamin monocatecholization. In this study, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 (P450) or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its antioxidant activity, whereas glucuronidation strongly reduces its antioxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. In contrast, in rat liver microsomes, catecholization is predominant over glucuronidation. In addition, rat liver produced two isomers of the glucuronide, whereas human liver produced only one glucuronide. These results suggest a significant species-based difference in the metabolism of sesamin between humans and rats. Kinetic studies using recombinant human UGT isoforms identified UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol. In addition, a good correlation was observed between the glucuronidation activity and UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes. These results strongly suggest that UGT2B7 plays an important role in glucuronidation of sesamin monocatechol. Interindividual difference among the 10 human liver microsomes is approximately 2-fold. These results, together with our previous results on the metabolism of sesamin by human P450, suggest a small interindividual difference in sesamin metabolism. We observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. On the basis of these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver.

To elucidate functional diversity of cytochrome P450 monooxygenases from the white-rot basidiomycete Phanerochaete chrysosporium (PcCYPs), we conducted a comprehensive functional screening using a wide variety of compounds. A functionomic survey resulted in characterization of novel PcCYP functions and discovery of versatile PcCYPs that exhibit broad substrate profiles. These results suggested that multifunctional properties of the versatile PcCYPs would play crucial roles in diversification of fungal metabolic systems involved in xenobiotic detoxification. To our knowledge, this is the first report describing multifunctional properties of versatile P450s from the fungal kingdom. An increased compilation of PcCYP functions will facilitate a thorough understanding of metabolic diversity in basidiomycetes and provide new insights that could also expedite practical applications in the biotechnology sector., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bioconversion processes, including specific hydroxylations, promise to be useful for practical applications because chemical syntheses often involve complex procedures. One of the successful applications of P450 reactions is the bioconversion of vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Recently, a cytochrome P450 gene encoding a vitamin D hydroxylase from the CYP107 family was cloned from Pseudonocardia autotrophica and is now applied in the bioconversion process that produces 1α,25-dihydroxyvitamin D₃. In addition, the directed evolution study of CYP107 has significantly enhanced its activity. On the other hand, we found that Streptomyces griseolus CYP105A1 can convert vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity. To date, multiple vitamin D hydroxylases have been found in bacteria, fungi, and mammals, suggesting that vitamin D is a popular substrate of the enzymes belonging to the P450 superfamily. A combination of these cytochrome P450s would produce a large number of compounds from vitamin D and its analogs. Therefore, we believe that the bioconversion of vitamin D and its analogs is one of the most promising P450 reactions in terms of practical application., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D(3) to its biologically active form, 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], suggesting the possibility for R73V/R84A to produce 1α,25(OH)(2)D(3). Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25(OH)(2)D(3) were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)(2)D(3), the second-step metabolite of vitamin D(3), was observed, although a considerable amount of vitamin D(3) still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)(3)D(3) and 1α,25(S),26(OH)(3)D(3), both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5:1. The crystal structure of the double variant with 1α,25(OH)(2)D(3) and a docking model of 1α,25(OH)(2)D(3) in its active site strongly suggest a hydrogen-bond network including the 1α-hydroxyl group, and several water molecules play an important role in the substrate-binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D(3) to produce biologically useful compounds., (© 2010 The Authors Journal compilation © 2010 FEBS.)

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min(-1)) and 2-MCDD (13 min(-1)) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichlorodibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzop-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.

We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of P. chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids and polycyclic aromatic hydrocarbons using the recombinant S. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyse 3'-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis-Menten equation but to Hill's equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular modelling of PcCYP50c and a docking study of flavone to its active site supported this hypothesis. The enzymatic properties of PcCYP50c and PcCYP142c resemble mammalian drug-metabolizing P450s, suggesting that their physiological roles are metabolism of xenobiotics. It is noted that these unique P. chrysosporium P450s have a potential for the production of useful flavonoids.

We previously revealed that Orf8 and Orf6, which were identified in the brassicicene C biosynthetic gene cluster in Alternaria brassicicola strain ATCC96836, were fusicoccadiene (FD) synthase and 16-O-methyltransferase, respectively. In the present Letter, the early biosynthetic steps after the formation of FD were investigated. Plasmids carrying the FD synthase gene, one (or two) of five cytochrome P450 genes (orf1, orf2, orf5, orf7, and orf11) identified in the cluster and a cytochrome P450 reductase gene cloned from strain ATCC96836 were constructed and introduced into Saccharomyces cerevisiae. Based on the structures of the compounds produced by the transformants, Orf1 is suggested to be an 8beta-hydroxylation enzyme that yields FD 8beta-ol (4), followed by 16-hydroxylation by Orf7 to produce FD 8beta16-diol (5).

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.3kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3'-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo-p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.

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.

Vitamin D 3 (VD 3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD 3 requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD 3 into 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) in two independent reactions, despite its low sequence identity with mammalian enzymes (<21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1alpha,25(OH) 2D 3. The compound 1alpha,25(OH) 2D 3 is positioned 11 A from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1-VD 3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B' and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1alpha-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD 3.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.

CYP27A1, CYP27B1, and CYP24A1 are members of the cytochrome P450 superfamily, and key enzymes of vitamin D(3) metabolism. CYP27A1 produced at least seven forms of minor metabolites including 1alpha, 25 (OH) (2)D(3) in addition to the major metabolite 25 (OH) D(3). In contrast, CYP27B1 appears to be a special enzyme to catalyze the hydroxylation at C-1alpha position of 25 (OH) D(3). Mutagenesis studies of CYP27B1 revealed the amino acid residues involved in substrate binding or interaction between CYP27B1 and adreno-doxin. On CYP24A1-dependent metabolism, remarkable metabolic processes of 1alpha, 25 (OH) (2)D(3) were observed. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha, 25 (OH) (2)D(3) into calcitroic acid. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha, 25 (OH) (2)D(3)-26, 23-lactone. Surprisingly, more than 70% of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1, and CYP24A1. In addition to the mitochondrial P450s, microsomal P450s also play important roles in vitamin D metabolism. Recent reports suggest that human microsomal CYP2R1 is more important than CYP27A1 as a vitamin D 25-hydroxylase. The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs appears to be due to the increased 1alpha, 25 (OH) (2)D(3) metabolism by CYP3A4. Some vitamin D analogs are good substrates for CYP3A1 while CYP24A1 is responsible for metabolism of most of vitamin D analogs.

The human genome project revealed a new member of the P450 family 2, CYP2W1, which has orthologous form in other vertebrate species, suggesting CYP2W1's significant physiological function. Recently, it was reported that CYP2W1 can metabolize arachidonic acid. In this study, we isolated human CYP2W1 cDNA, and successfully expressed truncated CYP2W1 lacking N-terminal 20 amino acids in Escherichia coli cells. In the bicistronic expression system for human CYP2W1 and NADPH-P450 reductase, the formation of blue pigment, indigo, was observed in bacterial cultures. Based on this result, we revealed that CYP2W1 catalyzes the oxidation of indole. In addition, CYP2W1 showed monooxygenase activity towards 3-methylindole and chlorzoxazone. However, no activity was observed towards fatty acids including arachidonic acid. Further analysis using an E. coli expression system will reveal substrate specificity of CYP2W1 and why this P450 isoform is universally conserved in vertebrates.

The decline in bone mineral density that occurs after long-term treatment with some antiepileptic drugs is thought to be mediated by increased vitamin D(3) metabolism. In this study, we show that the inducible enzyme CYP3A4 is a major source of oxidative metabolism of 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in human liver and small intestine and could contribute to this adverse effect. Heterologously-expressed CYP3A4 catalyzed the 23- and 24-hydroxylation of 1,25(OH)(2)D(3). No human microsomal cytochrome P450 enzyme tested, other than CYP3A5, supported these reactions. CYP3A4 exhibited opposite product stereochemical preference compared with that of CYP24A1, a known 1,25(OH)(2)D(3) hydroxylase. The three major metabolites generated by CYP3A4 were 1,23R,25(OH)(3)D(3), 1,24S,25(OH)(3)D(3), and 1,23S,25(OH)(3)D(3). Although the metabolic clearance of CYP3A4 was less than that of CYP24A1, comparison of metabolite profiles and experiments using CYP3A-specific inhibitors indicated that CYP3A4 was the dominant source of 1,25(OH)(2)D(3) 23- and 24-hydroxylase activity in both human small intestine and liver. Consistent with this observation, analysis of mRNA isolated from human intestine and liver (including samples from donors treated with phenytoin) revealed a general absence of CYP24A1 mRNA. In addition, expression of CYP3A4 mRNA in a panel of duodenal samples was significantly correlated with the mRNA level of a known vitamin D receptor gene target, calbindin-D9K. These and other data suggest that induction of CYP3A4-dependent 1,25(OH)(2)D(3) metabolism by antiepileptic drugs and other PXR ligands may diminish intestinal effects of the hormone and contribute to osteomalacia.

Only one isoform of cytochrome P450 (CYP) 2E subfamily was known in human and various animals. Three cDNAs corresponding to CYP 2E subfamily members (CYP2E-a, CYP2E-b and CYP2E-c) were obtained from feline liver. These cDNAs each had a 1488-bp nucleotide coding region encoding a predicted amino acid sequence of 495 residues. Eleven amino acid substitutions were observed between CYP2E-a and CYP2E-b, but only one substitution between CYP2E-b and CYP2E-c. The CO difference spectrums about 450 nm wave length and similar values of Vmax and Km of 6-hydoxygenase activity toward chlorzoxazone were observed in all three isoforms expressed in AH22 yeast cells. By PCR-RFLP, mRNA of the CYP2E-a was found to be expressed in liver, mononuclear cells, kidney, lung, stomach, intestine and pancreas, whereas CYP2E-b and CYP2E-c were expressed mainly in the liver and mononuclear cells. Expression of CYP2E-a was observed in the livers of all felines tested, but CYP2E-b and CYP2E-c were not expressed in all cats. The sequences of two different introns between exons I and II and between exons VII and VIII were obtained in genomic DNA from the feline liver. Based on these results, we conclude that cats have two highly similar CYP2E genes.

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5' portion (224-bp of 1st exon-8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg(-), Leu(-)), obtaining three good Arg(+) transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.

The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.

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 AL4V 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.

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.

Metabolism of polychlorinated dibenzo-p-dioxins by cytochrome P450 (P450) and UDP-glucuronosyltransferase (UGT) was examined using a recombinant enzyme system and human liver microsomes. We analyzed the glucuronidation of 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-triCDD) by rat CYP1A1 expressed in yeast microsomes and human UGT expressed in baculovirus-infected insect cells. Multiple UGT isozymes showed glucuronidation activity toward 8-hydroxy-2,3,7-triCDD (8-OH-2,3,7-triCDD), which was produced by CYP1A1. Of these UGTs, UGT1A1, 1A9, and 2B7, which are constitutively expressed in human livers, showed remarkable activity toward 8-OH-2,3,7-triCDD. The apparent kinetic parameters of glucuronidation, K(m) and k(cat), were estimated to be 0.8 microM and 1.8 min(-1), respectively, for UGT1A1, 0.8 microM and 1.8 min(-1), respectively, for UGT1A9, and 3.9 microM and 7.0 min(-1), respectively, for UGT2B7. In human liver microsomes with NADPH and UDP-glucuronic acid, 2,3,7-triCDD was first converted to 8-OH-2,3,7-triCDD, then further converted to its glucuronide. We compared the ability of 10 human liver microsomes to metabolize 2,3,7-triCDD and observed a significant difference in the glucuronidation of 2,3,7-triCDD that originated from the difference of the P450-dependent hydroxylation of 2,3,7-triCDD.

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.

Seventeen missense mutations of 25-hydroxyvitamin D(3) 1alpha-hydroxylase (CYP27B1) that cause Vitamin D-dependent rickets type I (VDDR-I) have been identified. To understand the mechanism by which each mutation disrupts 1alpha-hydroxylase activity and to visualize the substrate-binding site, we performed the homology modeling of CYP27B1. The three-dimensional (3D) structure of CYP27B1 was modeled on the basis of the crystal structure of rabbit CYP2C5, the first solved X-ray structure of a eukaryotic CYP. The 3D structure of CYP27B1 contains 17 helices and 6 beta-strands, and the overall structural folding is similar to the available structures of soluble CYPs as well as to the template CYP2C5. Mapping of the residues responsible for VDDR-I has provided much information concerning the function of each mutant. We have previously reported site-directed mutagenesis studies on several mutants of CYP27B1 causing VDDR-1, and suggested the role of each residue. All these suggestions are in good agreement with our 3D-model of CYP27B1. Furthermore, this model enabled us to predict the function of the other mutation residues responsible for VDDR-I.

Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).

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.

The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated. In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system. The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3). The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3). These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3). On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3). All of these metabolites showed considerable affinity for vitamin D receptor. These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes containing each of the human CYP. The metabolites of PCDDs were analyzed by HPLC and GC-MS. Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation with elimination of a chloride substituent. Although major CYPs in human liver such as CYP2C8, CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2 showed high catalytic activity toward DD and mono-, di-, and trichloroDDs. The kinetic parameters K(m)(app) and V(max) of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD) were estimated to be 0.30 microM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-triCDD was a good substrate for CYP1A1. However, none of the CYPs showed any detectable activity [<0.01 mol/min/mol of P450)] toward 2,3,7,8-tetraCDD. Substrate-induced absorption spectrum and inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity. It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability.