Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation

Xiao Yang He, Jian Shen, Wen Yu Hu, Xinxin Ding, Anthony Y.H. Lu, Jun Yan Hong

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val117→ Ala and Arg372→His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala117→Val and His372→ Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.

Original languageEnglish (US)
Pages (from-to)143-153
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume427
Issue number2
DOIs
StatePublished - Jul 15 2004
Externally publishedYes

Fingerprint

Hydroxylation
Arginine
Amino Acids
Valine
Alanine
Proteins
Amino Acid Substitution
Site-Directed Mutagenesis
Nicotine
Histidine
Carcinogens
Cytochrome P-450 Enzyme System
Tobacco
Amino Acid Sequence
Catalytic Domain
Mutagenesis
Kinetics
Substrates
Metabolism
coumarin 7

Keywords

  • Coumarin 7-hydroxylation
  • CYP2A13
  • CYP2A6
  • Cytochrome P450
  • Protein model
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation. / He, Xiao Yang; Shen, Jian; Hu, Wen Yu; Ding, Xinxin; Lu, Anthony Y.H.; Hong, Jun Yan.

In: Archives of Biochemistry and Biophysics, Vol. 427, No. 2, 15.07.2004, p. 143-153.

Research output: Contribution to journalArticle

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abstract = "Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5{\%} identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val117→ Ala and Arg372→His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala117→Val and His372→ Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.",
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T1 - Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation

AU - He, Xiao Yang

AU - Shen, Jian

AU - Hu, Wen Yu

AU - Ding, Xinxin

AU - Lu, Anthony Y.H.

AU - Hong, Jun Yan

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AB - Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val117→ Ala and Arg372→His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala117→Val and His372→ Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.

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KW - Site-directed mutagenesis

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