Limits...
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.

Show MeSH

Related in: MedlinePlus

Enzymatic activities of six CYP2D49 variants towards bufuralol.(A) Crystal structure of human CYP2D6. The conformation was constructed based on the crystallographic data of CYP2D6 (2F9Q) obtained from Protein Data Bank and drawn using Accelrys ViewerLite Version 5.0. Amino acid residues at positions 120, 216, 301, 481 and 483 are in ball-and-stick form. (B) Alignment of the partial amino acid sequences of human CYP2D6 and chicken CYP2D49. The number at the top is for human CYP2D6; the number at the bottom is for chicken CYP2D49. The arrowheads show the amino acid residues to be substituted. (C) Functional expression and detection of WT CYP2D49 and its variants. S9 fractions from Hela cells which were transfected with empty vector, pcDNA-CYP2D49, pcDNA-CYP2D49-V126A, V126F, E222A, D306A, F486A and F488A were extracted and separated on 10% SDS-PAGE gels. Western blotting analysis was then used to confirm that transient transfection had succeeded. Blots were probed with the anti-CYP2D49 antiserum and a β-actin antibody, respectively. (D) Bufuralol 1′-hydroxylation catalytic activities of six CYP2D49 variants. S9 fractions of Hela cells which were transfected with empty vector, WT CYP2D49 and six CYP2D49 mutants were incubated with bufuralol as described in “Materials and methods”. Metabolites produced in the reactions were analyzed by HPLC. The data are represented as nanomoles of metabolite/min/microgram of protein. The data shown are derived from a representative experiment reported as the mean (n = 3) ± SD. Differences between the WT and mutant protein samples are significant when ** p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3366968&req=5

pone-0038395-g005: Enzymatic activities of six CYP2D49 variants towards bufuralol.(A) Crystal structure of human CYP2D6. The conformation was constructed based on the crystallographic data of CYP2D6 (2F9Q) obtained from Protein Data Bank and drawn using Accelrys ViewerLite Version 5.0. Amino acid residues at positions 120, 216, 301, 481 and 483 are in ball-and-stick form. (B) Alignment of the partial amino acid sequences of human CYP2D6 and chicken CYP2D49. The number at the top is for human CYP2D6; the number at the bottom is for chicken CYP2D49. The arrowheads show the amino acid residues to be substituted. (C) Functional expression and detection of WT CYP2D49 and its variants. S9 fractions from Hela cells which were transfected with empty vector, pcDNA-CYP2D49, pcDNA-CYP2D49-V126A, V126F, E222A, D306A, F486A and F488A were extracted and separated on 10% SDS-PAGE gels. Western blotting analysis was then used to confirm that transient transfection had succeeded. Blots were probed with the anti-CYP2D49 antiserum and a β-actin antibody, respectively. (D) Bufuralol 1′-hydroxylation catalytic activities of six CYP2D49 variants. S9 fractions of Hela cells which were transfected with empty vector, WT CYP2D49 and six CYP2D49 mutants were incubated with bufuralol as described in “Materials and methods”. Metabolites produced in the reactions were analyzed by HPLC. The data are represented as nanomoles of metabolite/min/microgram of protein. The data shown are derived from a representative experiment reported as the mean (n = 3) ± SD. Differences between the WT and mutant protein samples are significant when ** p<0.01.

Mentions: Studying the influence of key amino acid residues on the catalytic activity of an enzyme may shed light on its catalytic mechanism. In human CYP2D6, amino acid residues such as Phe-120, Glu-216, Asp-301, Phe-481 and Phe-483 have been proposed to be involved in the substrate recognition and binding (Fig. 5A). To investigate whether the above five amino acid residues are also important for the bufuralol 1′-hydroxylation activity of CYP2D49, site-directed mutagenesis studies were conducted. We first aligned partial amino acid sequences of chicken CYP2D49 to that of human CYP2D6 and found that CYP2D49 owned the same amino acid residues as human CYP2D6 at all the sites except for the position 126, which is replaced by a valine residue (Fig. 5B). We then prepared plasmids encoding wild-type and mutant CYP2D49 (pcDNA-CYP2D49-V126A, V126F, E222A D306A, F486A and F488A) and transiently transfected them into Hela cells. Western blotting analysis was used to determine whether the transient transfection was successful. As shown in Fig. 5C, the transient transfection with WT and mutant CYP2D49 plasmids led to the significant over-expression of proteins with an estimated molecular mass of 55 kDa; these proteins were not detectable in control cells. The amount of CYP2D49 and its mutants detected was normalized against the detected amount of β-actin (data not shown). Furthermore, the bufuralol 1′-hydroxylation activities of the over-expressed WT and variant CYP2D49 proteins were determined by incubating bufuralol with S9 fractions from Hela cells which were transfected with empty vector, WT CYP2D49 and six CYP2D49 mutants. As shown in Fig. 5D, the WT CYP2D49 protein exhibited distinct bufuralol 1′-hydroxylation activity comparing to the control. However, significant decreases in the bufuralol 1′-hydroxylation activity were observed for all the variants, especially for the D306A variant (Fig. 5D). The bufuralol 1′-hydroxylation activities [V(nmol/min/mg protein)] of the WT CYP2D49 and the V126A, V126F, E222A, D306A, F486A and F488A variants were 309.77±6.14, 44.91±1.09, 18.17±0.53, 123.71±0.98, 2.07±0.70, 88.51±1.24 and 82.38±4.70 (mean ± SD), respectively, which indicates that these amino acid residues may play important roles in the bufuralol 1′-hydroxylation activity of chicken CYP2D49.


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)

Enzymatic activities of six CYP2D49 variants towards bufuralol.(A) Crystal structure of human CYP2D6. The conformation was constructed based on the crystallographic data of CYP2D6 (2F9Q) obtained from Protein Data Bank and drawn using Accelrys ViewerLite Version 5.0. Amino acid residues at positions 120, 216, 301, 481 and 483 are in ball-and-stick form. (B) Alignment of the partial amino acid sequences of human CYP2D6 and chicken CYP2D49. The number at the top is for human CYP2D6; the number at the bottom is for chicken CYP2D49. The arrowheads show the amino acid residues to be substituted. (C) Functional expression and detection of WT CYP2D49 and its variants. S9 fractions from Hela cells which were transfected with empty vector, pcDNA-CYP2D49, pcDNA-CYP2D49-V126A, V126F, E222A, D306A, F486A and F488A were extracted and separated on 10% SDS-PAGE gels. Western blotting analysis was then used to confirm that transient transfection had succeeded. Blots were probed with the anti-CYP2D49 antiserum and a β-actin antibody, respectively. (D) Bufuralol 1′-hydroxylation catalytic activities of six CYP2D49 variants. S9 fractions of Hela cells which were transfected with empty vector, WT CYP2D49 and six CYP2D49 mutants were incubated with bufuralol as described in “Materials and methods”. Metabolites produced in the reactions were analyzed by HPLC. The data are represented as nanomoles of metabolite/min/microgram of protein. The data shown are derived from a representative experiment reported as the mean (n = 3) ± SD. Differences between the WT and mutant protein samples are significant when ** p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038395-g005: Enzymatic activities of six CYP2D49 variants towards bufuralol.(A) Crystal structure of human CYP2D6. The conformation was constructed based on the crystallographic data of CYP2D6 (2F9Q) obtained from Protein Data Bank and drawn using Accelrys ViewerLite Version 5.0. Amino acid residues at positions 120, 216, 301, 481 and 483 are in ball-and-stick form. (B) Alignment of the partial amino acid sequences of human CYP2D6 and chicken CYP2D49. The number at the top is for human CYP2D6; the number at the bottom is for chicken CYP2D49. The arrowheads show the amino acid residues to be substituted. (C) Functional expression and detection of WT CYP2D49 and its variants. S9 fractions from Hela cells which were transfected with empty vector, pcDNA-CYP2D49, pcDNA-CYP2D49-V126A, V126F, E222A, D306A, F486A and F488A were extracted and separated on 10% SDS-PAGE gels. Western blotting analysis was then used to confirm that transient transfection had succeeded. Blots were probed with the anti-CYP2D49 antiserum and a β-actin antibody, respectively. (D) Bufuralol 1′-hydroxylation catalytic activities of six CYP2D49 variants. S9 fractions of Hela cells which were transfected with empty vector, WT CYP2D49 and six CYP2D49 mutants were incubated with bufuralol as described in “Materials and methods”. Metabolites produced in the reactions were analyzed by HPLC. The data are represented as nanomoles of metabolite/min/microgram of protein. The data shown are derived from a representative experiment reported as the mean (n = 3) ± SD. Differences between the WT and mutant protein samples are significant when ** p<0.01.
Mentions: Studying the influence of key amino acid residues on the catalytic activity of an enzyme may shed light on its catalytic mechanism. In human CYP2D6, amino acid residues such as Phe-120, Glu-216, Asp-301, Phe-481 and Phe-483 have been proposed to be involved in the substrate recognition and binding (Fig. 5A). To investigate whether the above five amino acid residues are also important for the bufuralol 1′-hydroxylation activity of CYP2D49, site-directed mutagenesis studies were conducted. We first aligned partial amino acid sequences of chicken CYP2D49 to that of human CYP2D6 and found that CYP2D49 owned the same amino acid residues as human CYP2D6 at all the sites except for the position 126, which is replaced by a valine residue (Fig. 5B). We then prepared plasmids encoding wild-type and mutant CYP2D49 (pcDNA-CYP2D49-V126A, V126F, E222A D306A, F486A and F488A) and transiently transfected them into Hela cells. Western blotting analysis was used to determine whether the transient transfection was successful. As shown in Fig. 5C, the transient transfection with WT and mutant CYP2D49 plasmids led to the significant over-expression of proteins with an estimated molecular mass of 55 kDa; these proteins were not detectable in control cells. The amount of CYP2D49 and its mutants detected was normalized against the detected amount of β-actin (data not shown). Furthermore, the bufuralol 1′-hydroxylation activities of the over-expressed WT and variant CYP2D49 proteins were determined by incubating bufuralol with S9 fractions from Hela cells which were transfected with empty vector, WT CYP2D49 and six CYP2D49 mutants. As shown in Fig. 5D, the WT CYP2D49 protein exhibited distinct bufuralol 1′-hydroxylation activity comparing to the control. However, significant decreases in the bufuralol 1′-hydroxylation activity were observed for all the variants, especially for the D306A variant (Fig. 5D). The bufuralol 1′-hydroxylation activities [V(nmol/min/mg protein)] of the WT CYP2D49 and the V126A, V126F, E222A, D306A, F486A and F488A variants were 309.77±6.14, 44.91±1.09, 18.17±0.53, 123.71±0.98, 2.07±0.70, 88.51±1.24 and 82.38±4.70 (mean ± SD), respectively, which indicates that these amino acid residues may play important roles in the bufuralol 1′-hydroxylation activity of chicken CYP2D49.

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.

Show MeSH
Related in: MedlinePlus