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Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49).

Cai H, Jiang J, Yang Q, Chen Q, Deng Y - PLoS ONE (2012)

Bottom Line: All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile.Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein.The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, People's Republic of China.

ABSTRACT
The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49) was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

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Phylogenetic tree of CYP2D amino acid sequences and genomic structures of human CYP2D6 and chicken CYP2D49.(A) Phylogeny of CYP2D amino acid sequences from the chicken and other animal species. The neighbor-joining tree was created using the Molecular Evolutionary Genetics Analysis Version 4 software. The numbers on the branches indicate the number of times per 100 bootstrap replicates that the branch appeared in the trees, estimated by a random resampling of the data. Only bootstrap values greater than 50% are shown. The scale bar represents 5 substitutions in 100 residues. (B) Genomic structures of human CYP2D6 and chicken CYP2D49. The diagram of the organization of the CYP2D subfamily in humans and chickens was determined by the BLAT analysis of the human and chicken genome data from NCBI database. Exons are indicated by boxes, whereas introns are indicated by lines. The lengths of the exons and introns are expressed in base pairs. The arrowheads indicate the direction of transcription.
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pone-0038395-g001: Phylogenetic tree of CYP2D amino acid sequences and genomic structures of human CYP2D6 and chicken CYP2D49.(A) Phylogeny of CYP2D amino acid sequences from the chicken and other animal species. The neighbor-joining tree was created using the Molecular Evolutionary Genetics Analysis Version 4 software. The numbers on the branches indicate the number of times per 100 bootstrap replicates that the branch appeared in the trees, estimated by a random resampling of the data. Only bootstrap values greater than 50% are shown. The scale bar represents 5 substitutions in 100 residues. (B) Genomic structures of human CYP2D6 and chicken CYP2D49. The diagram of the organization of the CYP2D subfamily in humans and chickens was determined by the BLAT analysis of the human and chicken genome data from NCBI database. Exons are indicated by boxes, whereas introns are indicated by lines. The lengths of the exons and introns are expressed in base pairs. The arrowheads indicate the direction of transcription.

Mentions: To identify the CYP2D isoforms in chickens, we first searched against the chicken EST database with the human CYP2D6 sequence. This resulted in the discovery of a unique unpublished cDNA clone (GenBank accession no. CR354312.1). Based on this discovery, we cloned the coding sequence of the cDNA (GenBank accession no. JQ241277) from the chicken liver for the first time. The sequence has been submitted to the Committee on P450 Nomenclature, which suggested the name CYP2D49 for this gene [25], [28]. To demonstrate the genetic distance between chicken CYP2D49 and other CYP2D isoforms in experimental animal species, the identities of the respective CYP2D isoforms are indicated as percentages in Table 1. The deduced 502 amino acid residues sequence of CYP2D49 possessed 52%–57% identities with the other CYP2D enzymes (Table 1). This finding, together with a phylogenetic comparison of the deduced amino acid sequence of CYP2D49 with the sequences of other CYP2D isoforms (Fig. 1A), indicated that chicken CYP2D49 belonged to the CYP2D family, but showed far evolutionary distance to other CYP2D enzymes because of the remarkable interspecies differences.


Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49).

Cai H, Jiang J, Yang Q, Chen Q, Deng Y - PLoS ONE (2012)

Phylogenetic tree of CYP2D amino acid sequences and genomic structures of human CYP2D6 and chicken CYP2D49.(A) Phylogeny of CYP2D amino acid sequences from the chicken and other animal species. The neighbor-joining tree was created using the Molecular Evolutionary Genetics Analysis Version 4 software. The numbers on the branches indicate the number of times per 100 bootstrap replicates that the branch appeared in the trees, estimated by a random resampling of the data. Only bootstrap values greater than 50% are shown. The scale bar represents 5 substitutions in 100 residues. (B) Genomic structures of human CYP2D6 and chicken CYP2D49. The diagram of the organization of the CYP2D subfamily in humans and chickens was determined by the BLAT analysis of the human and chicken genome data from NCBI database. Exons are indicated by boxes, whereas introns are indicated by lines. The lengths of the exons and introns are expressed in base pairs. The arrowheads indicate the direction of transcription.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366968&req=5

pone-0038395-g001: Phylogenetic tree of CYP2D amino acid sequences and genomic structures of human CYP2D6 and chicken CYP2D49.(A) Phylogeny of CYP2D amino acid sequences from the chicken and other animal species. The neighbor-joining tree was created using the Molecular Evolutionary Genetics Analysis Version 4 software. The numbers on the branches indicate the number of times per 100 bootstrap replicates that the branch appeared in the trees, estimated by a random resampling of the data. Only bootstrap values greater than 50% are shown. The scale bar represents 5 substitutions in 100 residues. (B) Genomic structures of human CYP2D6 and chicken CYP2D49. The diagram of the organization of the CYP2D subfamily in humans and chickens was determined by the BLAT analysis of the human and chicken genome data from NCBI database. Exons are indicated by boxes, whereas introns are indicated by lines. The lengths of the exons and introns are expressed in base pairs. The arrowheads indicate the direction of transcription.
Mentions: To identify the CYP2D isoforms in chickens, we first searched against the chicken EST database with the human CYP2D6 sequence. This resulted in the discovery of a unique unpublished cDNA clone (GenBank accession no. CR354312.1). Based on this discovery, we cloned the coding sequence of the cDNA (GenBank accession no. JQ241277) from the chicken liver for the first time. The sequence has been submitted to the Committee on P450 Nomenclature, which suggested the name CYP2D49 for this gene [25], [28]. To demonstrate the genetic distance between chicken CYP2D49 and other CYP2D isoforms in experimental animal species, the identities of the respective CYP2D isoforms are indicated as percentages in Table 1. The deduced 502 amino acid residues sequence of CYP2D49 possessed 52%–57% identities with the other CYP2D enzymes (Table 1). This finding, together with a phylogenetic comparison of the deduced amino acid sequence of CYP2D49 with the sequences of other CYP2D isoforms (Fig. 1A), indicated that chicken CYP2D49 belonged to the CYP2D family, but showed far evolutionary distance to other CYP2D enzymes because of the remarkable interspecies differences.

Bottom Line: All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile.Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein.The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, People's Republic of China.

ABSTRACT
The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49) was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

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