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The loss of enzyme activities by a single amino acid substitution of a newly cloned rabbit CYP2D isozyme, CYP2D24
International Congress Series 1233 (2002) 121 – 126
The loss of enzyme activities by a single amino acid substitution of a newly cloned rabbit CYP2D isozyme, CYP2D24 Mayumi Ishizuka, Yukio Yamamoto, Ayato Takada 1, Akio Kazusaka, Shoichi Fujita* Laboratory of Toxicology and Laboratory of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Abstract In the previous study, we cloned two novel cytochrome P450 (CYP) cDNAs (CYP2D23 and CYP2D24) from a rabbit liver cDNA library. CYP2D23 and CYP2D24 were heterogeneously expressed in 293T cells. CYP2D24 effectively catalyzed the oxidation of bufuralol and bunitrolol, the archetypal substrates of the CYP2D subfamily, while CYP2D23 exhibited catalytic activity only toward bufuralol. Furthermore, in this study, we isolated the variant of CYP2D24, in which 473 threonine was substituted with valine, and mutant CYP2D24 forms, in which 76lysine was replaced by proline. We determined the catalytic activities of the CYP2D24 forms and found that the mutants showed no activity related to drug metabolism. The computer-assisted analysis of the primary sequences of the CYP2D24 variant and mutant forms suggested that proline substitution results in alterations in the secondary structure and hydrophobicity of beta-Sheet 1, which was closed to Helix A, B and SRS-1, and was the proposed substrate-binding sites of P450. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; CYP2D24; Secondary structure; Hydrophobicity
1. Introduction Cytochrome P450 (CYP) is comprised of a superfamily of monooxygenases that catalyze metabolic activation and detoxification. The above can be found in a broad *
Corresponding author. Tel.: +81-11-706-6948; fax: +81-11-706-5105. E-mail address: [email protected] (S. Fujita). 1 Present address: Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 2 8 7 - X
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spectrum of structurally unrelated compounds including drugs, chemical carcinogens, environmental pollutants and endogenous substrates, such as steroids, fatty acids, vitamins, and prostanoids. The isozymes of the CYP2D subfamily play a role in the metabolism of a wide range of drugs, particularly cardiovascular agents and phychotropic agents. Our preliminary experiments on the rabbit liver, microsomal drug oxidation indicated that rabbits show catalytic activities towards bufuralol 1V-hydroxylation and bunitrolol 4hydroxylation, which are typical reactions for the CYP2D subfamily [1,2]. The catalytic activities suggest that there may be CYP2D isozymes expressed in rabbits. In a previous study, we successfully cloned two novel CYP2D isozymes, CYP2D23 and CYP2D24, from the liver of a male rabbit, using degenerate RT-PCR and rapid amplification of the cDNA ends (RACE) technique [3]. We determined the distribution of these isozymes in the rabbit’s organs and established their catalytic activities. In the process of CYP cloning, we isolated a variant and two PCR-mutant forms of CYP2D24, of which only one had an amino acid substitution (variant: 473threonine to valine, mutant: 76lysine to proline). In the current study, we are establishing the effects of the residue replacements on the catalytic activities, the secondary structures and the hydrophobicities of the CYP2D24 forms.
2. Materials and methods 2.1. Cloning and characterization of CYP2D24 wild, variant, and mutant forms CYP2D24 isoforms were separated from the cDNA library of a rabbit, as shown in the previous paper [3]. In the cloning process of CYP2D24, we obtained a variant and two mutant forms. The mutant of the CYP2D24 wild form, which was substituted from 76lysine to proline, was isolated due to a PCR error. We also replaced the same amino acid at the 76 residue in the CYP2D24 variant form. The 293T cells were transfected with the CYP2D24 clones and were maintained as shown in the previous study [3,4]. The levels of the CYP forms and their enzymatic activities were determined according to the published method [3]. 2.2. Analyses of secondary structure and hydrophobicity of CYP2D24 The secondary structure and hydrophobicity of CYP2D24 wild form, 473valine variant, and 76proline mutant forms were predicted using the structure prediction service of Parallel Protein Information Analysis (PAPIA) system (National Institute of Advanced Industrial Science and Technology, Computational Biology Research Center, Tokyo, Japan), and using the GENETYX program (Software Development, Tokyo, Japan). The structural analyses of the CYP forms using the PAPIA system were carried out according to the New Joint method [5].
3. Results and discussion In the previous study, we cloned two novel CYP cDNAs (CYP2D23 and CYP2D24) from a rabbit liver cDNA library. The open-reading frames of these cDNAs encode proteins,
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each of which are composed of 500 amino acids [3]. CYP2D23 mRNA was abundantly expressed in the liver and small intestine, but only slightly in the brain sections, whereas CYP2D24 mRNA was expressed in the liver, small intestine, and stomach. CYP2D23 and CYP2D24 were heterogeneously expressed in the 293T cells. CYP2D24 effectively catalyzed the oxidation of bufuralol and bunitrolol, which are the archetypal substrates of the CYP2D subfamily, while CYP2D23 exhibited catalytic activity exclusively towards bufuralol. Furthermore, we cloned a variant and two PCR-mutant forms of CYP2D24, which had only one amino acid substitution as follows; variant, 473threonine to valine: PCR-mutants, 76 lysine to proline of CYP2D24 wild and variant forms. The variant form of CYP2D24 had high metabolic activity of the bunitrolol 4-hydroxylation and bufuralol 1V-hydroxylation (Fig. 1). The result of the PAPIA system was that the secondary structure was not changed in the CYP2D24 variant form. The secondary structure of the heme-binding region, where it was closed to residue 473, was conserved in variant form (Fig. 2). In the present study, remarkable changes in CYP-enzymatic activities were observed in the PCR-mutated CYP2D24 forms, which substituted only one amino acid at 76 residues. In bunitrolol and bufuralol hydroxylations, no metabolic activity of these mutant forms of CYP2D24 wild and variant was observed (Fig. 1). We suggest that the mutations at the 76 residues changed the structure of the functional domain in CYP2D24. The amino acids at the 76 residues in the CYP2D subfamilies (CYP2D1, CYP2D2, CYP2D6, CYP2D9, CYP2D14, CYP2D15, CYP2D16, CYP2D17, CYP2D23 and CYP2D25) are lysine, isoleusine, methionine, glutamine, and phenylalanine (Figs. 2 and 3). The results of the
Fig. 1. Metabolic activities of CYP2D24 chimera. T(W):CYP2D24 wild form, V(W):variant ( 473threonine to valine) of wild form, T(P) and V(P): 76lysine to proline of CYP2D24 wild and variant forms, respectively, MOCK: vector.
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Fig. 2. A schematic diagram of the predicted secondary structure of CYP2D24.
Fig. 3. Sequence alignment of the CYP2D subfamilies.
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Fig. 4. Predicted secondary structure information and hydropathy index of CYP2D24.
prediction analyses for the secondary structure suggested that substitutions at the 76 residues to these amino acids had no effect on the structure of CYP2D24. However, proline could possibly have the ability to cause the conformation change of CYP2D24, which is probably due to the change in the beta-turn motif and the Helix B structure (Figs. 2 and 3). Furthermore, the hydrophobicities of beta Sheet-1 of CYP2D24 were altered in the proline-substituted forms (Fig. 4). Thus, we concluded that the deficiency of enzymatic activity of the 76proline CYP2D24s was due to a structural change in the N-terminal domain, including beta-Sheet-1, Helix B and SRS-1, proposed substrate-binding sites of CYP.
Acknowledgements We thank Prof. J. Miyazaki, Osaka University Medical School, for kindly providing the pCAGGS expression vector.
References [1] T. Suzuki, S. Narimatsu, S. Fujita, Y. Masubuchi, S. Umeda, S. Imaoka, Y. Funae, Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats, Drug Metab. Dispos. 20 (3) (1992) 367 – 373. [2] Y. Yamamoto, T. Tasaki, A. Nakamura, H. Iwata, A. Kazusaka, F.J. Gonzalez, S. Fujita, Molecular basis of the Dark Agouti rat drug oxidation polymorphism: importance of CYP2D1 and CYP2D2, Pharmacogenetics 8 (1) (1998) 73 – 82. [3] Y. Yamamoto, M. Ishizuka, A. Takada, S. Fujita, Cloning, tissue distribution, and functional expression of
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two novel rabbit cytochrome P450 isozymes, CYP2D23 and CYP2D24, J. Biochem. (Tokyo) 124 (3) (1998) 503 – 508. [4] H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector, Gene 108 (2) (1991) 193 – 199. [5] Y. Akiyama, K. Onizuka, T. Noguchi, M. Ando, Parallel protein information analysis (PAPIA) system running on a 64-node PC cluster, Genome Inf. Ser. Workshop Genome Inf. 9 (1998) 131 – 140.
The loss of enzyme activities by a single amino acid substitution of a newly cloned rabbit CYP2D isozyme, CYP2D24 Mayumi Ishizuka, Yukio Yamamoto, Ayato Takada 1, Akio Kazusaka, Shoichi Fujita* Laboratory of Toxicology and Laboratory of Microbiology, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan
Abstract In the previous study, we cloned two novel cytochrome P450 (CYP) cDNAs (CYP2D23 and CYP2D24) from a rabbit liver cDNA library. CYP2D23 and CYP2D24 were heterogeneously expressed in 293T cells. CYP2D24 effectively catalyzed the oxidation of bufuralol and bunitrolol, the archetypal substrates of the CYP2D subfamily, while CYP2D23 exhibited catalytic activity only toward bufuralol. Furthermore, in this study, we isolated the variant of CYP2D24, in which 473 threonine was substituted with valine, and mutant CYP2D24 forms, in which 76lysine was replaced by proline. We determined the catalytic activities of the CYP2D24 forms and found that the mutants showed no activity related to drug metabolism. The computer-assisted analysis of the primary sequences of the CYP2D24 variant and mutant forms suggested that proline substitution results in alterations in the secondary structure and hydrophobicity of beta-Sheet 1, which was closed to Helix A, B and SRS-1, and was the proposed substrate-binding sites of P450. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; CYP2D24; Secondary structure; Hydrophobicity
1. Introduction Cytochrome P450 (CYP) is comprised of a superfamily of monooxygenases that catalyze metabolic activation and detoxification. The above can be found in a broad *
Corresponding author. Tel.: +81-11-706-6948; fax: +81-11-706-5105. E-mail address: [email protected] (S. Fujita). 1 Present address: Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 2 8 7 - X
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spectrum of structurally unrelated compounds including drugs, chemical carcinogens, environmental pollutants and endogenous substrates, such as steroids, fatty acids, vitamins, and prostanoids. The isozymes of the CYP2D subfamily play a role in the metabolism of a wide range of drugs, particularly cardiovascular agents and phychotropic agents. Our preliminary experiments on the rabbit liver, microsomal drug oxidation indicated that rabbits show catalytic activities towards bufuralol 1V-hydroxylation and bunitrolol 4hydroxylation, which are typical reactions for the CYP2D subfamily [1,2]. The catalytic activities suggest that there may be CYP2D isozymes expressed in rabbits. In a previous study, we successfully cloned two novel CYP2D isozymes, CYP2D23 and CYP2D24, from the liver of a male rabbit, using degenerate RT-PCR and rapid amplification of the cDNA ends (RACE) technique [3]. We determined the distribution of these isozymes in the rabbit’s organs and established their catalytic activities. In the process of CYP cloning, we isolated a variant and two PCR-mutant forms of CYP2D24, of which only one had an amino acid substitution (variant: 473threonine to valine, mutant: 76lysine to proline). In the current study, we are establishing the effects of the residue replacements on the catalytic activities, the secondary structures and the hydrophobicities of the CYP2D24 forms.
2. Materials and methods 2.1. Cloning and characterization of CYP2D24 wild, variant, and mutant forms CYP2D24 isoforms were separated from the cDNA library of a rabbit, as shown in the previous paper [3]. In the cloning process of CYP2D24, we obtained a variant and two mutant forms. The mutant of the CYP2D24 wild form, which was substituted from 76lysine to proline, was isolated due to a PCR error. We also replaced the same amino acid at the 76 residue in the CYP2D24 variant form. The 293T cells were transfected with the CYP2D24 clones and were maintained as shown in the previous study [3,4]. The levels of the CYP forms and their enzymatic activities were determined according to the published method [3]. 2.2. Analyses of secondary structure and hydrophobicity of CYP2D24 The secondary structure and hydrophobicity of CYP2D24 wild form, 473valine variant, and 76proline mutant forms were predicted using the structure prediction service of Parallel Protein Information Analysis (PAPIA) system (National Institute of Advanced Industrial Science and Technology, Computational Biology Research Center, Tokyo, Japan), and using the GENETYX program (Software Development, Tokyo, Japan). The structural analyses of the CYP forms using the PAPIA system were carried out according to the New Joint method [5].
3. Results and discussion In the previous study, we cloned two novel CYP cDNAs (CYP2D23 and CYP2D24) from a rabbit liver cDNA library. The open-reading frames of these cDNAs encode proteins,
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each of which are composed of 500 amino acids [3]. CYP2D23 mRNA was abundantly expressed in the liver and small intestine, but only slightly in the brain sections, whereas CYP2D24 mRNA was expressed in the liver, small intestine, and stomach. CYP2D23 and CYP2D24 were heterogeneously expressed in the 293T cells. CYP2D24 effectively catalyzed the oxidation of bufuralol and bunitrolol, which are the archetypal substrates of the CYP2D subfamily, while CYP2D23 exhibited catalytic activity exclusively towards bufuralol. Furthermore, we cloned a variant and two PCR-mutant forms of CYP2D24, which had only one amino acid substitution as follows; variant, 473threonine to valine: PCR-mutants, 76 lysine to proline of CYP2D24 wild and variant forms. The variant form of CYP2D24 had high metabolic activity of the bunitrolol 4-hydroxylation and bufuralol 1V-hydroxylation (Fig. 1). The result of the PAPIA system was that the secondary structure was not changed in the CYP2D24 variant form. The secondary structure of the heme-binding region, where it was closed to residue 473, was conserved in variant form (Fig. 2). In the present study, remarkable changes in CYP-enzymatic activities were observed in the PCR-mutated CYP2D24 forms, which substituted only one amino acid at 76 residues. In bunitrolol and bufuralol hydroxylations, no metabolic activity of these mutant forms of CYP2D24 wild and variant was observed (Fig. 1). We suggest that the mutations at the 76 residues changed the structure of the functional domain in CYP2D24. The amino acids at the 76 residues in the CYP2D subfamilies (CYP2D1, CYP2D2, CYP2D6, CYP2D9, CYP2D14, CYP2D15, CYP2D16, CYP2D17, CYP2D23 and CYP2D25) are lysine, isoleusine, methionine, glutamine, and phenylalanine (Figs. 2 and 3). The results of the
Fig. 1. Metabolic activities of CYP2D24 chimera. T(W):CYP2D24 wild form, V(W):variant ( 473threonine to valine) of wild form, T(P) and V(P): 76lysine to proline of CYP2D24 wild and variant forms, respectively, MOCK: vector.
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Fig. 2. A schematic diagram of the predicted secondary structure of CYP2D24.
Fig. 3. Sequence alignment of the CYP2D subfamilies.
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Fig. 4. Predicted secondary structure information and hydropathy index of CYP2D24.
prediction analyses for the secondary structure suggested that substitutions at the 76 residues to these amino acids had no effect on the structure of CYP2D24. However, proline could possibly have the ability to cause the conformation change of CYP2D24, which is probably due to the change in the beta-turn motif and the Helix B structure (Figs. 2 and 3). Furthermore, the hydrophobicities of beta Sheet-1 of CYP2D24 were altered in the proline-substituted forms (Fig. 4). Thus, we concluded that the deficiency of enzymatic activity of the 76proline CYP2D24s was due to a structural change in the N-terminal domain, including beta-Sheet-1, Helix B and SRS-1, proposed substrate-binding sites of CYP.
Acknowledgements We thank Prof. J. Miyazaki, Osaka University Medical School, for kindly providing the pCAGGS expression vector.
References [1] T. Suzuki, S. Narimatsu, S. Fujita, Y. Masubuchi, S. Umeda, S. Imaoka, Y. Funae, Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol 4-hydroxylation in liver microsomes of male rats, Drug Metab. Dispos. 20 (3) (1992) 367 – 373. [2] Y. Yamamoto, T. Tasaki, A. Nakamura, H. Iwata, A. Kazusaka, F.J. Gonzalez, S. Fujita, Molecular basis of the Dark Agouti rat drug oxidation polymorphism: importance of CYP2D1 and CYP2D2, Pharmacogenetics 8 (1) (1998) 73 – 82. [3] Y. Yamamoto, M. Ishizuka, A. Takada, S. Fujita, Cloning, tissue distribution, and functional expression of
126
M. Ishizuka et al. / International Congress Series 1233 (2002) 121–126
two novel rabbit cytochrome P450 isozymes, CYP2D23 and CYP2D24, J. Biochem. (Tokyo) 124 (3) (1998) 503 – 508. [4] H. Niwa, K. Yamamura, J. Miyazaki, Efficient selection for high-expression transfectants with a novel eukaryotic vector, Gene 108 (2) (1991) 193 – 199. [5] Y. Akiyama, K. Onizuka, T. Noguchi, M. Ando, Parallel protein information analysis (PAPIA) system running on a 64-node PC cluster, Genome Inf. Ser. Workshop Genome Inf. 9 (1998) 131 – 140.