Review Received: 13 April 2014
Revised: 15 June 2014
Accepted article published: 31 July 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.3871
Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants Hideo Ohkawa* and Hideyuki Inui Abstract A yeast gene expression system originally established for mammalian cytochrome P450 monooxygenase cDNAs was applied to functional analysis of a number of mammalian and plant P450 species, including 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). The human P450 species CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 were identified as P450 species metabolising various agrochemicals and environmental chemicals. CYP2C9 and CYP2E1 specifically metabolised sulfonylurea herbicides and halogenated hydrocarbons respectively. Plant P450 species metabolising phenylurea and sulfonylurea herbicides were also identified mainly as the CYP71 family, although CYP76B1, CYP81B1 and CYP81B2 metabolised phenylurea herbicides. The transgenic plants expressing these mammalian and plant P450 species were applied to herbicide tolerance as well as phytoremediation of agrochemical and environmental chemical residues. The combined use of CYP1A1, CYP2B6 and CYP2C19 belonging to two families and three subfamilies covered a wide variety of herbicide tolerance and phytoremediation of these residues. The use of 2,4-D-and bromoxynil-induced CYP71AH11 in tobacco seemed to enhance herbicide tolerance and selectivity. © 2014 Society of Chemical Industry Keywords: cytochrome P450; NADPH-cytochrome P450 oxidoreductase; agrochemical; environmental chemical; herbicide; metabolism; tolerance; phytoremediation
1
INTRODUCTION
Molecular mechanisms of metabolism of agrochemicals and related environmental chemicals are important for understanding biodegradability, tolerance and toxicity to target and non-target organisms. Most lipophilic foreign chemicals are taken up into mammalian and plant cells by a passive diffusion mechanism. In phase I metabolism, the chemicals undergo metabolic reactions mainly catalysed by cytochrome P450 (P450 or CYP) monooxygenases, glutathione S-transferases and esterases. In phase II, many metabolites are conjugated with hydrophilic materials, followed by transfer to cell compartments and excretion from cells. Recent advances made in biochemical and genetic researches have clarified the roles and function of enzyme systems involved in metabolism of foreign chemicals. In particular, gene engineering of P450 species in mammals and plants has clarified their functions and created novel opportunities for their application in herbicide tolerance and bioremediation of agrochemical and environmental chemical residues.1,2
2 GENE ENGINEERING OF MAMMALIAN P450 SPECIES P450 monooxygenases consisting of many P450 species and one or two NADPH-cytochrome P450 oxidoreductases (P450 reductases) located on the endoplasmic reticulum in mammalian cells are involved in the metabolism of a wide variety of foreign chemicals. These P450 species catalyse various oxidation reactions and Pest Manag Sci (2014)
contribute to detoxification or activation of foreign chemicals in mammals. The first attempt to clone a mammalian P450 cDNA was reported in 1982 by Fujii-Kuriyama et al.3 The cDNA sequence revealed the amino acid sequence of rat CYP2B1. In 1984, Yabusaki et al.4 reported cDNA cloning and the primary structure of rat CYP1A1 (former P450c), although the P450 species CYP1A1 was later named by Nebert et al.5 Thereafter, cDNA clones and the primary structures of a large number of P450 species were reported as listed in the cytochrome P450 homepage (http://drnelson.uthsc.edu/CytochromeP450.html).6 In 1985, Oeda et al.7 attempted to express rat CYP1A1 cDNA in the yeast Saccharomyces cerevisiae AH22. The CYP1A1 produced in the yeast cells was localised on microsomes and exhibited CYP1A1-dependent monooxygenase activity by interaction with endogenous yeast P450 reductase. This is the first report on heterologous expression of a mammalian P450 cDNA. Thereafter, Sakaki et al.8 reported the functional expression of chimeric P450 genes constructed between rat CYP1A1 and CYP1A2 cDNAs in the yeast. They suggested that the central area of the P450 sequences contributed to the determination of substrate specificity. Furthermore, rat CYP1A1 and P450 reductase fused enzyme
∗
Correspondence to: Hideo Ohkawa, Kobe University, 14–14 Kashiodai, Kita-ku, Kobe, Hyogo 651–1255, Japan. E-mail: [email protected] Research Centre for Environmental Genomics, Kobe University, Kobe, Hyogo, Japan
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www.soci.org genes were constructed and expressed in the yeast.9 The fused enzyme produced in the yeast cells exhibited higher specific activity than the combination of rat CYP1A1 and P450 reductase enzymes. In addition, Lacour and Ohkawa10 reported that rat CYP1A1, maize ferredoxin and pea ferredoxin-NADP+ reductase fused enzymes were produced on microsomes of the yeast cells and showed CYP1A1-dependent monooxygenase activity. Then, the yeast gene expression system was practically utilised for functional analysis of various P450 species, characterisation of novel P450 species and construction of engineered P450 monooxygenases.
3 METABOLISM OF AGROCHEMICALS AND RELATED ENVIRONMENTAL CHEMICALS IN MAMMALIAN P450 SPECIES Microsomal P450 species are responsible for the phase I metabolism of a large number of foreign chemicals in mammals. It was suggested that 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) cover more than 90% of P450-dependent metabolism of foreign chemicals.11 These P450 species belong to three families and seven subfamilies. Therefore, these P450 species were characterised for metabolism of a wide variety of agrochemicals and related environmental chemicals. These P450 cDNAs were each expressed in the yeast cells. The microsomal fraction prepared from the recombinant yeast cells was examined for metabolism of a number of lipophilic chemicals. As shown in Table 1, the insecticide methoxychlor was metabolised by seven P450 species, including CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18 and CYP2C19, which catalysed O-demethylation. The herbicide esprocarb was metabolised by five P450 species, including CYP1A1, CYP1A2, CYP2B6, CYP2C19 and CYP2D6. These five P450s catalysed alkyl and ring hydroxylations. The herbicide chlorotoluron was also metabolised by four P450 species, including CYP1A1, CYP1A2, CYP2C19 and CYP2D6. These four P450s mediated both N-demethylation and ring-methyl hydroxylation. The herbicide atrazine was metabolised by CYP1A1, CYP1A2, CYP2C19 and CYP2D6 catalysing both N-deethylation and N-deisopropylation. The herbicide simetryn was easily metabolised by CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 catalysing N-deethylation, and also by CYP1A1, CYP1A2, CYP2C9 and CYP2C19 catalysing S-demethylation. These chemicals seemed to be quite biodegradable. On the other hand, the sulfonylurea herbicides chlorsulfuron, imazosulfuron and triasulfuron were specifically metabolised by CYP2C9, which catalysed ring hydroxylation. The herbicide quizalofop-ethyl was also specifically metabolised by CYP1A1 catalysing ring hydroxylation. CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 metabolised 16, 17, 15, 13 and 22 chemicals respectively among 33 compounds tested. These P450 species seemed to be multifunctional and showed broad and overlapping substrate specificity. CYP2E1 was a unique enzyme, which specifically metabolised the halogenated hydrocarbons trichloroethylene and ethylene dibromide.12 CYP2A6 catalysed N-deethylation and desulfuration towards simazine and fenitrothion respectively. Therefore, both CYP2E1 and CYP2A6 showed limited substrate specificity towards 33 chemicals tested. Human CYP1A1 hardly metabolised 3,3′ ,4,4′ ,5-pentachlorobiphenyl, although rat CYP1A1 metabolised the chemical to give 4-OH-3,3′ ,4′ , 5-tetrachlorobiphenyl and 4-OH-3,3′ ,4′ ,5,5′ -pentachlorobiphenyl.
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H Ohkawa, H Inui
The CYP1A1 species from human and rat showed differences in the metabolism of the compound.13 Based on the results listed in Table 1, objective P450 species are able to be chosen for the purpose of metabolism of certain agrochemicals and environmental chemicals.
4 METABOLISM OF HERBICIDES IN PLANT P450 SPECIES Plant P450s metabolised herbicides and determined herbicide tolerance and selectivity. Also, P450-dependent metabolism contributed to characterisation of residues of agrochemicals and environmental chemicals. However, molecular characterisation of such P450 species was quite limited.14,15 As shown in Table 2, the first attempt to isolate a plant P450 species metabolising herbicide was reported in 1998 by Cabello-Hurtado et al.16 CYP81B1 in Jerusalem artichoke was found to be involved in metabolism of the phenylurea herbicide chlorotoluron and to catalyse ring-methyl hydroxylation. In 1999, Siminszky et al.17 reported that soybean CYP71A10 catalysed both N-demethylation and ring-methyl hydroxylation towards chlorotoluron, and N-demethylation towards fluometuron, linuron and diuron. Also, Yamada et al.18 reported in 2000 that CYP71AH11 (former CYP71A11) and CYP81B2 in tobacco induced by 2,4-D catalysed both N-demethylation and ring-methyl hydroxylation towards chlorotoluron. Didierjean et al.19 reported in 2002 that CYP76B1 in Jerusalem artichoke metabolised chlorotoluron and isoproturon through N-demethylation. Then, Tsujii et al.20 reported that CYP71R4 in Lolium rigidum biotype W1R2, which is resistant to phenylurea and triazine herbicides, catalysed N-demethylation and ring-methyl hydroxylation towards chlorotoluron. Therefore, CYP71R4 seemed in part to be involved in herbicide tolerance of the biotype WLR2. RNA-seq transcription analyses revealed the presence of the CYP72A subfamily related to herbicide diclofop resistance in L. rigidum, as reported by Gaines et al.21 Furthermore, Xiang et al.22 reported that wheat CYP71C6v1 catalysed ring hydroxylation towards the sulfonylurea herbicides chlorsulfuron and triasulfuron. Based on the results listed in Table 2, the CYP71 family was mainly involved in metabolism of phenylurea and sulfonylurea herbicides, although CYP76B1, CYP81B1 and CYP81B2 also metabolised phenylurea herbicides. It was found that tobacco CYP71AH11 induced by 2,4-D was more actively induced by the herbicide bromoxynil.23 Han et al.24 reported that 2,4-D protected L. rigidum against the herbicide diclofop-methyl owing to enhanced herbicide metabolism. Thus, both 2,4-D and bromoxynil seem to be useful for enhancement of herbicide tolerance and selectivity.
5 ENGINEERED MAMMALIAN AND PLANT P450 GENES FOR HERBICIDE TOLERANCE AND PHYTOREMEDIATION P450 monooxygenases in plants contributed to determination of herbicide tolerance and selectivity as well as characterisation of agrochemical and environmental chemical residues. Certain P450 species in mammals and plants were selected on the basis of their molecular characterisation and applied to herbicide tolerance and phytoremediation.25,26 As shown in Table 3, the first attempt to engineer herbicide metabolism and tolerance into plants was performed with rat CYP1A1 and yeast P450 reductase fused genes in 1994 by Shiota et al.27 The transgenic tobacco plants
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Metabolism of agro-and environmental chemicals by P450s
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Table 1. Metabolism of agrochemicals and related environmental chemicals in 11 human P450 species11,12 and rat CYP1A113 Chemical
Reaction
P450 species
Alachlor Atrazine Benfuresate
N-Demethoxymethylation N-Deethylation and N-deisopropylation Alkyl and ring hydroxylation
Chlorsulfuron Chlorotoluron
Ring hydroxylation N-Demethylation and ring-methyl hydroxylation N-Demethylation
Diuron
CYP1A1, CYP1A2, CYP2C9, CYP2C18, CYP2C19 and CYP2D6 CYP1A1, CYP1A2, CYP2C9 and CYP2D6 CYP1A1, CYP1A2, CYP2B6, CYP2C19 and CYP3A4 CYP2B6 and CYP2C19 CYP2C9 CYP2B6 and CYP2C19 CYP1A1, CYP1A2 and CYP2C19 CYP2B6, CYP2C18 and CYP2C19 CYP1A1, CYP1A2, CYP2C18 and CY2C19 CYP1A1 and CYP3A4 CYP2C19 CYP1A1, CYP2B6 and CYP2C19 CYP2C8, CYP2C9, CYP2C18 and CYP2C19 CYP1A1, CYP1A2, CYP2C8 and CYP3A4 CYP1A1 CYP1A1, CYP1A2, CYP2A6, CYP2C19 and CYP2D6 CYP1A1, CYP1A2, CYP2C9 and CYP2C19 CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 CYP2C9 CYP1A1, CYP2C8, CYP2C18 and CYP2D6 CYP1A1, CYP1A2, CYP2B6 and CYP2C19 CYP2A6, CYP2B6, CYP2C18, CYP2C19 and CYP3A4 CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 CYP2B6 and CYP2C19 CYP1A2, CYP2B6 and CYP2C19 CYP2E1 CYP2E1 Rat CYP1A1
Ring hydroxylation Alkyl and ring hydroxylation O-Deethylation Ring hydroxylation N-Demethylation Ring hydroxylation O-Demethylation N-Demethylation Alkyl and ring hydroxylation O-Demethylation Cleavage of thiocarbamate O-Demethylation Alkyl and ring hydroxylation Ring hydroxylation N-Deethylation S-Demethylation N-Deethylation
Esprocarb Ethofumesate Imazosulfuron Mefenacet Metolachlor Norflurazon Pyrazoxyfen Pyributicarb Pyriminobac-methyl Quizalofop-ethyl Simazine Simetryn
Triasulfuron Azinphos-methyl Carbaryl Fenitrothion Methoxychlor
Ring hydroxylation Desulfuration Ring hydroxylation Desulfuration O-Demethylation
4-Nonylphenol 4-n-Nonylphenol Trichloroethylene Ethylene dibromide 3,3′ ,4,4′ ,5-Pentachlorobiphenyl
Alkyl and ring hydroxylation Alkyl and ring hydroxylation Oxidation of vinyl Debromination 4Cl → OH
CYP2B6 and CYP2C18 CYP1A1, CYP1A2, CYP2C19 and CYP2D6 CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6 and CYP3A4 CYP2C9 CYP1A1, CYP1A2, CYP2C19 and CYP2D6
Table 2. Plant P450 species metabolising herbicides P450 species
Plant
CYP71A1017
Soybean
CYP71AH1118 CYP71C6v122 CYP71R420 CYP76B119 CYP81B116 CYP81B218
Tobacco Wheat Lolium rigidum Jerusalem artichoke Jerusalem artichoke Tobacco
Reaction N-Demethylation and ring-methyl hydroxylation N-Demethylation N-Demethylation and ring-methyl hydroxylation Ring hydroxylation N-Demethylation and ring-methyl hydroxylation N-Demethylation Ring-methyl hydroxylation N-Demethylation and ring-methyl hydroxylation
expressing the CYP1A1 and P450 reductase fused enzyme gene showed enhanced metabolism of the herbicide chlorotoluron and herbicide tolerance. Soybean CYP71A10 gene was expressed in tobacco plants and shown to confer increased herbicide tolerance to chlorotoluron and linuron.17 As human CYP1A1, CYP2B6 and CYP2C19 exhibited a broad and overlapping substrate specificity, the combination of these three P450 species seemed to cover Pest Manag Sci (2014)
Herbicide Chlorotoluron Fluometuron, linuron and diuron Chlorotoluron Chlorsulfuron and triasulfuron Chlorotoluron Chlorotoluron and isoproturon Chlorotoluron Chlorotoluron
metabolism of various herbicides and to show cross-tolerance to these herbicides. These three P450 cDNAs were individually and simultaneously expressed in potato plants. The transgenic potato plants expressing three P450 cDNAs simultaneously more actively metabolised chlorotoluron, atrazine, pyributicarb and methoxychlor as compared with the single P450-expressing plants. Thus, the combination of these three P450 species seemed
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H Ohkawa, H Inui
Table 3. Transgenic plants harbouring P450 genes for herbicide tolerance and phytoremediation P450 species
Plant
Rat CYP1A1/yeast P450 reductase fused enzyme27
Tobacco
Soybean CYP71A1017 Human CYP1A1, CYP2B6 and/or CYP2C1911,28
Tobacco Potato
Human CYP2E112
Tobacco
Human CYP2C929
Rice
Human CYP2C1929
Rice
Jerusalem artichoke CYP76B119 Human CYP2B630
Tobacco and Arabidopsis Rice
Human CYP1A1, CYP2B6 and CYP2C1931 Indica rice CYP72A3132
Rice Arabidopsis
to function synergistically in metabolism of the chemicals.11,28 On the other hand, Doty et al.12 reported that the transgenic tobacco plants expressing human CYP2E1 cDNA metabolised the halogenated hydrocarbons trichloroethylene and ethylene dibromide, which contaminated groundwater. The transgenic rice plants expressing human CYP2C9 metabolised the sulfonylurea herbicides chlorsulfuron and imazosulfuron and exhibited tolerance to these herbicides.29 Also, human CYP2C19-expressing rice plants showed cross-tolerance to the herbicides chlorsulfuron, mefenacet, metolachlor, norflurazon and pyributicarb.29 The CYP2B6-expressing rice plants grown in soils under paddy field conditions showed tolerance to the herbicides alachlor and metolachlor. The CYP2B6 rice plants were confirmed to metabolise metolachlor and to remove much higher amounts of metolachlor residues in soils under paddy field conditions.30 The rice plants coexpressing human CYP1A1, CYP2B6 and CYP2C19 were more tolerant to the herbicides atrazine, metolachlor and norflurazon with different herbicide actions than non-transgenic rice plants, owing to increased metabolism of these herbicides.31 The gene of CYP72A31 isolated from indica rice, which shows tolerance to the herbicide bispyribac sodium, was expressed in Arabidopsis and showed tolerance to the herbicide bensulfuron-methyl, suggesting that CYP72A31 may metabolise bispyribac sodium and bensulfuron-methyl. The CYP72A31 gene was used as a selectable marker of plant transformation.32 As observed with mammalian and plant P450 species listed in Table 3, these P450 species enhanced herbicide tolerance and phytoremediation of agrochemical and environmental chemical residues.
6
Metabolism and/or tolerance
CONCLUDING REMARKS
Gene engineering of P450 species metabolising foreign chemicals in mammals and plants was established for herbicide tolerance and phytoremediation of agrochemical and environmental chemical residues. The combination of human CYP1A1, CYP2B6 and CYP2C19 in different families and subfamilies has metabolised a wide variety of agrochemicals and environmental chemicals, and shown cross-tolerance to various herbicides owing to their synergistic activity. Human CYP2C9 specifically metabolising the sulfonylurea herbicides has been used for herbicide
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Metabolism of chlorotoluron, tolerance to chlorotoluron Tolerance to linuron and chlortoluron Metabolism of atrazine, tolerance to atrazine, acetochlor, metolachlor, chlortoluron, methabenzthiazuron, norflurazon and pyributicarb Metabolism of trichloroethylene and ethylene dibromide Metabolism of chlorsulfuron and imazosulfuron, tolerance to chlorsulfuron and mefenacet Tolerance to chlorsulfuron, mefenacet, metolachlor, norflurazon and pyributicarb Tolerance to chlorotoluron, isoproturon and linuron Metabolism of metolachlor, tolerance to alachlor and metolachlor Tolerance to atrazine, metolachlor and norflurazon Tolerance to bensulfuron-methyl
tolerance in rice plants.29 Human CYP2E1 metabolising halogenated hydrocarbons has been utilised for phytoremediation of these contaminants.12 On the other hand, CYP1A1 and CYP1A2 are inducible by 2,3,7,8-tetrachlorodibonzo-p-dioxin and certain polychlorinated biphenyl congeners, which are ligands for an aryl hydrocarbon receptor (AhR). The AhR ligands increase CYP1A1and CYP1A2-dependent metabolism of foreign chemicals in mammals, resulting in enhancement of detoxification and/or activation of these chemicals.2 The Arabidopsis genome is estimated to contain as many as 286 different P450 genes. cDNA microarray analysis indicated that a full-length cDNA collection contains 49 different P450 genes, which are induced by both abiotic and biotic stresses,33 but does not contain the P450 species listed in Table 2. On the other hand, CYP71AH11 metabolising the herbicide chlorotoluron has been induced in tobacco plants by treatment with the herbicides 2,4-D and bromoxynil, probably owing to the generation of reactive oxygen species.23 In addition, 2,4-D has enhanced the metabolism of the herbicide diclofop-methyl in L. rigidum and protected the susceptible biotype against the herbicide.24 Therefore, 2,4-D and bromoxynil seem to be useful for enhancement of herbicide tolerance and selectivity.
REFERENCES 1 Inui H and Ohkawa H, Herbicide resistance in transgenic plants with mammalian P450 monooxygenase genes. Pest Manag Sci 61:286–291 (2005). 2 Shimazu S, Inui H and Ohkawa H, Phytomonitoring and phytoremediation of agrochemical and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs). J Agric Food Chem 59:2870–2875 (2011). 3 Fujii-Kuriyama Y, Mizukami Y, Kawajiri K, Segawa K and Muramatsu M, Primary structure of a cytochrome P450: coding nucleotide sequence of phenobarbital-inducible cytochrome P450 cDNA from rat liver. Proc Natl Acad Sci USA 79:2793–2797 (1982). 4 Yabusaki Y, Shimizu M, Murakami H, Nakamura K, Oeda K and Ohkawa H, Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P450MC. Nucleic Acids Res 12:2929–2938 (1984). 5 Nebert DW, Nelson DR, Adesnik M, Coon MJ, Estabrook RW, Gonzalez FJ, et al, The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci. DNA 8:1–13 (1989).
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Metabolism of agro-and environmental chemicals by P450s 6 Nelson DR, The Cytochrome P450 Homepage. [Online]. Available: http://drnelson.uthsc.edu/CytochromeP450.html [18 August 2014]. 7 Oeda K, Sakaki T and Ohkawa H, Expression of rat liver cytochrome P-450MC cDNA in Saccharomyces cerevisiae. DNA 4:203–210 (1985). 8 Sakaki T, Shibata M, Yabusaki Y and Ohkawa H, Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d. DNA 6:31–39 (1987). 9 Murakami H, Yabusaki Y, Sakaki T, Shibata M and Ohkawa H, A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase. DNA 6:189–197 (1987). 10 Lacour T and Ohkawa H, Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP+ reductase from plant chloroplasts. Biochim Biophys Acta 1433:87–102 (1999). 11 Inui H, Shiota N, Motoi Y, Ido Y, Inoue T, Kodama T, et al, Metabolism of herbicides and other chemicals in human cytochrome P450 species and in transgenic potato plants co-expressing human CYP1A1, CYP2B6 and CYP2C19. J Pestic Sci 26:28–40 (2001). 12 Doty SL, Shang TQ, Wilson AM, Tangen J, Westerngreen AD, Newman LA, et al, Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1. Proc Natl Acad Sci USA 97:6287–6291 (2000). 13 Yamazaki K, Suzuki M, Itoh T, Yamamoto K, Kanemitsu M, Matsumura C, et al, Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3′ ,4,4′ ,5-pentachlorobiphenyl. J Biochem 149:487–494 (2011). 14 Siminszky B, Plant cytochrome P450-mediated herbicide metabolism. Plantchem Rev 5:445–458 (2006). 15 Powles SB and Yu Q, Evolution in action: plant resistance to herbicides. Annu Rev Plant Biol 61:317–347 (2010). 16 Cabello-Hurtado F, Batard Y, Salaun JP, Durst F, Pinot F and Werck-Reichhart D, Cloning, expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that catalyzes in-chain hydroxylation of fatty acids. J Biol Chem 273:7260–7267 (1998). 17 Siminszky B, Corbin FT, Ward ER, Fleischmann TJ and Dewey RE, Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides. Proc Natl Acad Sci USA 96:1750–1755 (1999). 18 Yamada T, Kambara Y, Imaishi H and Ohkawa H, Molecular cloning of novel cytochrome P450 species induced by chemical treatments in cultured tobacco cells. Pestic Biochem Physiol 68:11–25 (2000). 19 Didierjean L, Gondet L, Perkins R, Lau SMC, Schaller H, O’Keefe DP, et al, Engineering herbicide metabolism in tobacco and Arabidopsis with CYP76B1, a cytochrome P450 enzyme from Jerusalem artichoke. Plant Physiol 130:179–189 (2002). 20 Tsujii H, Dillon N and Ohkawa H, Molecular functions of cytochrome P450 species involved in herbicide resistance in Lolium rigidum biotype WLR2. Abstracts of Papers of 7th International
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Revised: 15 June 2014
Accepted article published: 31 July 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/ps.3871
Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants Hideo Ohkawa* and Hideyuki Inui Abstract A yeast gene expression system originally established for mammalian cytochrome P450 monooxygenase cDNAs was applied to functional analysis of a number of mammalian and plant P450 species, including 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). The human P450 species CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 were identified as P450 species metabolising various agrochemicals and environmental chemicals. CYP2C9 and CYP2E1 specifically metabolised sulfonylurea herbicides and halogenated hydrocarbons respectively. Plant P450 species metabolising phenylurea and sulfonylurea herbicides were also identified mainly as the CYP71 family, although CYP76B1, CYP81B1 and CYP81B2 metabolised phenylurea herbicides. The transgenic plants expressing these mammalian and plant P450 species were applied to herbicide tolerance as well as phytoremediation of agrochemical and environmental chemical residues. The combined use of CYP1A1, CYP2B6 and CYP2C19 belonging to two families and three subfamilies covered a wide variety of herbicide tolerance and phytoremediation of these residues. The use of 2,4-D-and bromoxynil-induced CYP71AH11 in tobacco seemed to enhance herbicide tolerance and selectivity. © 2014 Society of Chemical Industry Keywords: cytochrome P450; NADPH-cytochrome P450 oxidoreductase; agrochemical; environmental chemical; herbicide; metabolism; tolerance; phytoremediation
1
INTRODUCTION
Molecular mechanisms of metabolism of agrochemicals and related environmental chemicals are important for understanding biodegradability, tolerance and toxicity to target and non-target organisms. Most lipophilic foreign chemicals are taken up into mammalian and plant cells by a passive diffusion mechanism. In phase I metabolism, the chemicals undergo metabolic reactions mainly catalysed by cytochrome P450 (P450 or CYP) monooxygenases, glutathione S-transferases and esterases. In phase II, many metabolites are conjugated with hydrophilic materials, followed by transfer to cell compartments and excretion from cells. Recent advances made in biochemical and genetic researches have clarified the roles and function of enzyme systems involved in metabolism of foreign chemicals. In particular, gene engineering of P450 species in mammals and plants has clarified their functions and created novel opportunities for their application in herbicide tolerance and bioremediation of agrochemical and environmental chemical residues.1,2
2 GENE ENGINEERING OF MAMMALIAN P450 SPECIES P450 monooxygenases consisting of many P450 species and one or two NADPH-cytochrome P450 oxidoreductases (P450 reductases) located on the endoplasmic reticulum in mammalian cells are involved in the metabolism of a wide variety of foreign chemicals. These P450 species catalyse various oxidation reactions and Pest Manag Sci (2014)
contribute to detoxification or activation of foreign chemicals in mammals. The first attempt to clone a mammalian P450 cDNA was reported in 1982 by Fujii-Kuriyama et al.3 The cDNA sequence revealed the amino acid sequence of rat CYP2B1. In 1984, Yabusaki et al.4 reported cDNA cloning and the primary structure of rat CYP1A1 (former P450c), although the P450 species CYP1A1 was later named by Nebert et al.5 Thereafter, cDNA clones and the primary structures of a large number of P450 species were reported as listed in the cytochrome P450 homepage (http://drnelson.uthsc.edu/CytochromeP450.html).6 In 1985, Oeda et al.7 attempted to express rat CYP1A1 cDNA in the yeast Saccharomyces cerevisiae AH22. The CYP1A1 produced in the yeast cells was localised on microsomes and exhibited CYP1A1-dependent monooxygenase activity by interaction with endogenous yeast P450 reductase. This is the first report on heterologous expression of a mammalian P450 cDNA. Thereafter, Sakaki et al.8 reported the functional expression of chimeric P450 genes constructed between rat CYP1A1 and CYP1A2 cDNAs in the yeast. They suggested that the central area of the P450 sequences contributed to the determination of substrate specificity. Furthermore, rat CYP1A1 and P450 reductase fused enzyme
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Correspondence to: Hideo Ohkawa, Kobe University, 14–14 Kashiodai, Kita-ku, Kobe, Hyogo 651–1255, Japan. E-mail: [email protected] Research Centre for Environmental Genomics, Kobe University, Kobe, Hyogo, Japan
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www.soci.org genes were constructed and expressed in the yeast.9 The fused enzyme produced in the yeast cells exhibited higher specific activity than the combination of rat CYP1A1 and P450 reductase enzymes. In addition, Lacour and Ohkawa10 reported that rat CYP1A1, maize ferredoxin and pea ferredoxin-NADP+ reductase fused enzymes were produced on microsomes of the yeast cells and showed CYP1A1-dependent monooxygenase activity. Then, the yeast gene expression system was practically utilised for functional analysis of various P450 species, characterisation of novel P450 species and construction of engineered P450 monooxygenases.
3 METABOLISM OF AGROCHEMICALS AND RELATED ENVIRONMENTAL CHEMICALS IN MAMMALIAN P450 SPECIES Microsomal P450 species are responsible for the phase I metabolism of a large number of foreign chemicals in mammals. It was suggested that 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) cover more than 90% of P450-dependent metabolism of foreign chemicals.11 These P450 species belong to three families and seven subfamilies. Therefore, these P450 species were characterised for metabolism of a wide variety of agrochemicals and related environmental chemicals. These P450 cDNAs were each expressed in the yeast cells. The microsomal fraction prepared from the recombinant yeast cells was examined for metabolism of a number of lipophilic chemicals. As shown in Table 1, the insecticide methoxychlor was metabolised by seven P450 species, including CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18 and CYP2C19, which catalysed O-demethylation. The herbicide esprocarb was metabolised by five P450 species, including CYP1A1, CYP1A2, CYP2B6, CYP2C19 and CYP2D6. These five P450s catalysed alkyl and ring hydroxylations. The herbicide chlorotoluron was also metabolised by four P450 species, including CYP1A1, CYP1A2, CYP2C19 and CYP2D6. These four P450s mediated both N-demethylation and ring-methyl hydroxylation. The herbicide atrazine was metabolised by CYP1A1, CYP1A2, CYP2C19 and CYP2D6 catalysing both N-deethylation and N-deisopropylation. The herbicide simetryn was easily metabolised by CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 catalysing N-deethylation, and also by CYP1A1, CYP1A2, CYP2C9 and CYP2C19 catalysing S-demethylation. These chemicals seemed to be quite biodegradable. On the other hand, the sulfonylurea herbicides chlorsulfuron, imazosulfuron and triasulfuron were specifically metabolised by CYP2C9, which catalysed ring hydroxylation. The herbicide quizalofop-ethyl was also specifically metabolised by CYP1A1 catalysing ring hydroxylation. CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 metabolised 16, 17, 15, 13 and 22 chemicals respectively among 33 compounds tested. These P450 species seemed to be multifunctional and showed broad and overlapping substrate specificity. CYP2E1 was a unique enzyme, which specifically metabolised the halogenated hydrocarbons trichloroethylene and ethylene dibromide.12 CYP2A6 catalysed N-deethylation and desulfuration towards simazine and fenitrothion respectively. Therefore, both CYP2E1 and CYP2A6 showed limited substrate specificity towards 33 chemicals tested. Human CYP1A1 hardly metabolised 3,3′ ,4,4′ ,5-pentachlorobiphenyl, although rat CYP1A1 metabolised the chemical to give 4-OH-3,3′ ,4′ , 5-tetrachlorobiphenyl and 4-OH-3,3′ ,4′ ,5,5′ -pentachlorobiphenyl.
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The CYP1A1 species from human and rat showed differences in the metabolism of the compound.13 Based on the results listed in Table 1, objective P450 species are able to be chosen for the purpose of metabolism of certain agrochemicals and environmental chemicals.
4 METABOLISM OF HERBICIDES IN PLANT P450 SPECIES Plant P450s metabolised herbicides and determined herbicide tolerance and selectivity. Also, P450-dependent metabolism contributed to characterisation of residues of agrochemicals and environmental chemicals. However, molecular characterisation of such P450 species was quite limited.14,15 As shown in Table 2, the first attempt to isolate a plant P450 species metabolising herbicide was reported in 1998 by Cabello-Hurtado et al.16 CYP81B1 in Jerusalem artichoke was found to be involved in metabolism of the phenylurea herbicide chlorotoluron and to catalyse ring-methyl hydroxylation. In 1999, Siminszky et al.17 reported that soybean CYP71A10 catalysed both N-demethylation and ring-methyl hydroxylation towards chlorotoluron, and N-demethylation towards fluometuron, linuron and diuron. Also, Yamada et al.18 reported in 2000 that CYP71AH11 (former CYP71A11) and CYP81B2 in tobacco induced by 2,4-D catalysed both N-demethylation and ring-methyl hydroxylation towards chlorotoluron. Didierjean et al.19 reported in 2002 that CYP76B1 in Jerusalem artichoke metabolised chlorotoluron and isoproturon through N-demethylation. Then, Tsujii et al.20 reported that CYP71R4 in Lolium rigidum biotype W1R2, which is resistant to phenylurea and triazine herbicides, catalysed N-demethylation and ring-methyl hydroxylation towards chlorotoluron. Therefore, CYP71R4 seemed in part to be involved in herbicide tolerance of the biotype WLR2. RNA-seq transcription analyses revealed the presence of the CYP72A subfamily related to herbicide diclofop resistance in L. rigidum, as reported by Gaines et al.21 Furthermore, Xiang et al.22 reported that wheat CYP71C6v1 catalysed ring hydroxylation towards the sulfonylurea herbicides chlorsulfuron and triasulfuron. Based on the results listed in Table 2, the CYP71 family was mainly involved in metabolism of phenylurea and sulfonylurea herbicides, although CYP76B1, CYP81B1 and CYP81B2 also metabolised phenylurea herbicides. It was found that tobacco CYP71AH11 induced by 2,4-D was more actively induced by the herbicide bromoxynil.23 Han et al.24 reported that 2,4-D protected L. rigidum against the herbicide diclofop-methyl owing to enhanced herbicide metabolism. Thus, both 2,4-D and bromoxynil seem to be useful for enhancement of herbicide tolerance and selectivity.
5 ENGINEERED MAMMALIAN AND PLANT P450 GENES FOR HERBICIDE TOLERANCE AND PHYTOREMEDIATION P450 monooxygenases in plants contributed to determination of herbicide tolerance and selectivity as well as characterisation of agrochemical and environmental chemical residues. Certain P450 species in mammals and plants were selected on the basis of their molecular characterisation and applied to herbicide tolerance and phytoremediation.25,26 As shown in Table 3, the first attempt to engineer herbicide metabolism and tolerance into plants was performed with rat CYP1A1 and yeast P450 reductase fused genes in 1994 by Shiota et al.27 The transgenic tobacco plants
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Metabolism of agro-and environmental chemicals by P450s
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Table 1. Metabolism of agrochemicals and related environmental chemicals in 11 human P450 species11,12 and rat CYP1A113 Chemical
Reaction
P450 species
Alachlor Atrazine Benfuresate
N-Demethoxymethylation N-Deethylation and N-deisopropylation Alkyl and ring hydroxylation
Chlorsulfuron Chlorotoluron
Ring hydroxylation N-Demethylation and ring-methyl hydroxylation N-Demethylation
Diuron
CYP1A1, CYP1A2, CYP2C9, CYP2C18, CYP2C19 and CYP2D6 CYP1A1, CYP1A2, CYP2C9 and CYP2D6 CYP1A1, CYP1A2, CYP2B6, CYP2C19 and CYP3A4 CYP2B6 and CYP2C19 CYP2C9 CYP2B6 and CYP2C19 CYP1A1, CYP1A2 and CYP2C19 CYP2B6, CYP2C18 and CYP2C19 CYP1A1, CYP1A2, CYP2C18 and CY2C19 CYP1A1 and CYP3A4 CYP2C19 CYP1A1, CYP2B6 and CYP2C19 CYP2C8, CYP2C9, CYP2C18 and CYP2C19 CYP1A1, CYP1A2, CYP2C8 and CYP3A4 CYP1A1 CYP1A1, CYP1A2, CYP2A6, CYP2C19 and CYP2D6 CYP1A1, CYP1A2, CYP2C9 and CYP2C19 CYP1A1, CYP1A2, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 CYP2C9 CYP1A1, CYP2C8, CYP2C18 and CYP2D6 CYP1A1, CYP1A2, CYP2B6 and CYP2C19 CYP2A6, CYP2B6, CYP2C18, CYP2C19 and CYP3A4 CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18 and CYP2C19 CYP2B6 and CYP2C19 CYP1A2, CYP2B6 and CYP2C19 CYP2E1 CYP2E1 Rat CYP1A1
Ring hydroxylation Alkyl and ring hydroxylation O-Deethylation Ring hydroxylation N-Demethylation Ring hydroxylation O-Demethylation N-Demethylation Alkyl and ring hydroxylation O-Demethylation Cleavage of thiocarbamate O-Demethylation Alkyl and ring hydroxylation Ring hydroxylation N-Deethylation S-Demethylation N-Deethylation
Esprocarb Ethofumesate Imazosulfuron Mefenacet Metolachlor Norflurazon Pyrazoxyfen Pyributicarb Pyriminobac-methyl Quizalofop-ethyl Simazine Simetryn
Triasulfuron Azinphos-methyl Carbaryl Fenitrothion Methoxychlor
Ring hydroxylation Desulfuration Ring hydroxylation Desulfuration O-Demethylation
4-Nonylphenol 4-n-Nonylphenol Trichloroethylene Ethylene dibromide 3,3′ ,4,4′ ,5-Pentachlorobiphenyl
Alkyl and ring hydroxylation Alkyl and ring hydroxylation Oxidation of vinyl Debromination 4Cl → OH
CYP2B6 and CYP2C18 CYP1A1, CYP1A2, CYP2C19 and CYP2D6 CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6 and CYP3A4 CYP2C9 CYP1A1, CYP1A2, CYP2C19 and CYP2D6
Table 2. Plant P450 species metabolising herbicides P450 species
Plant
CYP71A1017
Soybean
CYP71AH1118 CYP71C6v122 CYP71R420 CYP76B119 CYP81B116 CYP81B218
Tobacco Wheat Lolium rigidum Jerusalem artichoke Jerusalem artichoke Tobacco
Reaction N-Demethylation and ring-methyl hydroxylation N-Demethylation N-Demethylation and ring-methyl hydroxylation Ring hydroxylation N-Demethylation and ring-methyl hydroxylation N-Demethylation Ring-methyl hydroxylation N-Demethylation and ring-methyl hydroxylation
expressing the CYP1A1 and P450 reductase fused enzyme gene showed enhanced metabolism of the herbicide chlorotoluron and herbicide tolerance. Soybean CYP71A10 gene was expressed in tobacco plants and shown to confer increased herbicide tolerance to chlorotoluron and linuron.17 As human CYP1A1, CYP2B6 and CYP2C19 exhibited a broad and overlapping substrate specificity, the combination of these three P450 species seemed to cover Pest Manag Sci (2014)
Herbicide Chlorotoluron Fluometuron, linuron and diuron Chlorotoluron Chlorsulfuron and triasulfuron Chlorotoluron Chlorotoluron and isoproturon Chlorotoluron Chlorotoluron
metabolism of various herbicides and to show cross-tolerance to these herbicides. These three P450 cDNAs were individually and simultaneously expressed in potato plants. The transgenic potato plants expressing three P450 cDNAs simultaneously more actively metabolised chlorotoluron, atrazine, pyributicarb and methoxychlor as compared with the single P450-expressing plants. Thus, the combination of these three P450 species seemed
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H Ohkawa, H Inui
Table 3. Transgenic plants harbouring P450 genes for herbicide tolerance and phytoremediation P450 species
Plant
Rat CYP1A1/yeast P450 reductase fused enzyme27
Tobacco
Soybean CYP71A1017 Human CYP1A1, CYP2B6 and/or CYP2C1911,28
Tobacco Potato
Human CYP2E112
Tobacco
Human CYP2C929
Rice
Human CYP2C1929
Rice
Jerusalem artichoke CYP76B119 Human CYP2B630
Tobacco and Arabidopsis Rice
Human CYP1A1, CYP2B6 and CYP2C1931 Indica rice CYP72A3132
Rice Arabidopsis
to function synergistically in metabolism of the chemicals.11,28 On the other hand, Doty et al.12 reported that the transgenic tobacco plants expressing human CYP2E1 cDNA metabolised the halogenated hydrocarbons trichloroethylene and ethylene dibromide, which contaminated groundwater. The transgenic rice plants expressing human CYP2C9 metabolised the sulfonylurea herbicides chlorsulfuron and imazosulfuron and exhibited tolerance to these herbicides.29 Also, human CYP2C19-expressing rice plants showed cross-tolerance to the herbicides chlorsulfuron, mefenacet, metolachlor, norflurazon and pyributicarb.29 The CYP2B6-expressing rice plants grown in soils under paddy field conditions showed tolerance to the herbicides alachlor and metolachlor. The CYP2B6 rice plants were confirmed to metabolise metolachlor and to remove much higher amounts of metolachlor residues in soils under paddy field conditions.30 The rice plants coexpressing human CYP1A1, CYP2B6 and CYP2C19 were more tolerant to the herbicides atrazine, metolachlor and norflurazon with different herbicide actions than non-transgenic rice plants, owing to increased metabolism of these herbicides.31 The gene of CYP72A31 isolated from indica rice, which shows tolerance to the herbicide bispyribac sodium, was expressed in Arabidopsis and showed tolerance to the herbicide bensulfuron-methyl, suggesting that CYP72A31 may metabolise bispyribac sodium and bensulfuron-methyl. The CYP72A31 gene was used as a selectable marker of plant transformation.32 As observed with mammalian and plant P450 species listed in Table 3, these P450 species enhanced herbicide tolerance and phytoremediation of agrochemical and environmental chemical residues.
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Metabolism and/or tolerance
CONCLUDING REMARKS
Gene engineering of P450 species metabolising foreign chemicals in mammals and plants was established for herbicide tolerance and phytoremediation of agrochemical and environmental chemical residues. The combination of human CYP1A1, CYP2B6 and CYP2C19 in different families and subfamilies has metabolised a wide variety of agrochemicals and environmental chemicals, and shown cross-tolerance to various herbicides owing to their synergistic activity. Human CYP2C9 specifically metabolising the sulfonylurea herbicides has been used for herbicide
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Metabolism of chlorotoluron, tolerance to chlorotoluron Tolerance to linuron and chlortoluron Metabolism of atrazine, tolerance to atrazine, acetochlor, metolachlor, chlortoluron, methabenzthiazuron, norflurazon and pyributicarb Metabolism of trichloroethylene and ethylene dibromide Metabolism of chlorsulfuron and imazosulfuron, tolerance to chlorsulfuron and mefenacet Tolerance to chlorsulfuron, mefenacet, metolachlor, norflurazon and pyributicarb Tolerance to chlorotoluron, isoproturon and linuron Metabolism of metolachlor, tolerance to alachlor and metolachlor Tolerance to atrazine, metolachlor and norflurazon Tolerance to bensulfuron-methyl
tolerance in rice plants.29 Human CYP2E1 metabolising halogenated hydrocarbons has been utilised for phytoremediation of these contaminants.12 On the other hand, CYP1A1 and CYP1A2 are inducible by 2,3,7,8-tetrachlorodibonzo-p-dioxin and certain polychlorinated biphenyl congeners, which are ligands for an aryl hydrocarbon receptor (AhR). The AhR ligands increase CYP1A1and CYP1A2-dependent metabolism of foreign chemicals in mammals, resulting in enhancement of detoxification and/or activation of these chemicals.2 The Arabidopsis genome is estimated to contain as many as 286 different P450 genes. cDNA microarray analysis indicated that a full-length cDNA collection contains 49 different P450 genes, which are induced by both abiotic and biotic stresses,33 but does not contain the P450 species listed in Table 2. On the other hand, CYP71AH11 metabolising the herbicide chlorotoluron has been induced in tobacco plants by treatment with the herbicides 2,4-D and bromoxynil, probably owing to the generation of reactive oxygen species.23 In addition, 2,4-D has enhanced the metabolism of the herbicide diclofop-methyl in L. rigidum and protected the susceptible biotype against the herbicide.24 Therefore, 2,4-D and bromoxynil seem to be useful for enhancement of herbicide tolerance and selectivity.
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Metabolism of agro-and environmental chemicals by P450s 6 Nelson DR, The Cytochrome P450 Homepage. [Online]. Available: http://drnelson.uthsc.edu/CytochromeP450.html [18 August 2014]. 7 Oeda K, Sakaki T and Ohkawa H, Expression of rat liver cytochrome P-450MC cDNA in Saccharomyces cerevisiae. DNA 4:203–210 (1985). 8 Sakaki T, Shibata M, Yabusaki Y and Ohkawa H, Expression in Saccharomyces cerevisiae of chimeric cytochrome P450 cDNAs constructed from cDNAs for rat cytochrome P450c and P450d. DNA 6:31–39 (1987). 9 Murakami H, Yabusaki Y, Sakaki T, Shibata M and Ohkawa H, A genetically engineered P450 monooxygenase: construction of the functional fused enzyme between rat cytochrome P450c and NADPH-cytochrome P450 reductase. DNA 6:189–197 (1987). 10 Lacour T and Ohkawa H, Engineering and biochemical characterization of the rat microsomal cytochrome P4501A1 fused to ferredoxin and ferredoxin-NADP+ reductase from plant chloroplasts. Biochim Biophys Acta 1433:87–102 (1999). 11 Inui H, Shiota N, Motoi Y, Ido Y, Inoue T, Kodama T, et al, Metabolism of herbicides and other chemicals in human cytochrome P450 species and in transgenic potato plants co-expressing human CYP1A1, CYP2B6 and CYP2C19. J Pestic Sci 26:28–40 (2001). 12 Doty SL, Shang TQ, Wilson AM, Tangen J, Westerngreen AD, Newman LA, et al, Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1. Proc Natl Acad Sci USA 97:6287–6291 (2000). 13 Yamazaki K, Suzuki M, Itoh T, Yamamoto K, Kanemitsu M, Matsumura C, et al, Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3′ ,4,4′ ,5-pentachlorobiphenyl. J Biochem 149:487–494 (2011). 14 Siminszky B, Plant cytochrome P450-mediated herbicide metabolism. Plantchem Rev 5:445–458 (2006). 15 Powles SB and Yu Q, Evolution in action: plant resistance to herbicides. Annu Rev Plant Biol 61:317–347 (2010). 16 Cabello-Hurtado F, Batard Y, Salaun JP, Durst F, Pinot F and Werck-Reichhart D, Cloning, expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that catalyzes in-chain hydroxylation of fatty acids. J Biol Chem 273:7260–7267 (1998). 17 Siminszky B, Corbin FT, Ward ER, Fleischmann TJ and Dewey RE, Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides. Proc Natl Acad Sci USA 96:1750–1755 (1999). 18 Yamada T, Kambara Y, Imaishi H and Ohkawa H, Molecular cloning of novel cytochrome P450 species induced by chemical treatments in cultured tobacco cells. Pestic Biochem Physiol 68:11–25 (2000). 19 Didierjean L, Gondet L, Perkins R, Lau SMC, Schaller H, O’Keefe DP, et al, Engineering herbicide metabolism in tobacco and Arabidopsis with CYP76B1, a cytochrome P450 enzyme from Jerusalem artichoke. Plant Physiol 130:179–189 (2002). 20 Tsujii H, Dillon N and Ohkawa H, Molecular functions of cytochrome P450 species involved in herbicide resistance in Lolium rigidum biotype WLR2. Abstracts of Papers of 7th International
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