Characterization of marmoset CYP2B6

CDNA cloning, protein expression and enzymatic functions

Kei Mayumi, Nobumitsu Hanioka, Kazufumi Masuda, Akiko Koeda, Shinsaku Naito, Atsuro Miyata, Shizuo Narimatsu

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.

Original languageEnglish
Pages (from-to)1182-1194
Number of pages13
JournalBiochemical Pharmacology
Volume85
Issue number8
DOIs
Publication statusPublished - Apr 15 2013

Fingerprint

efavirenz
Callithrix
Cloning
Bupropion
Organism Cloning
Liver
Proteins
Mixed Function Oxygenases
Amino Acids
Oxidation
Enzymes
Macaca fascicularis
Hydroxylation
Kinetics
Insects
Substrates
Metabolism
Pharmaceutical Preparations
Cytochrome P-450 CYP2B6
Substitution reactions

Keywords

  • Bupropion
  • cDNA cloning
  • Docking simulation
  • Efavirenz
  • Marmoset CYP2B6

ASJC Scopus subject areas

  • Pharmacology
  • Biochemistry

Cite this

Mayumi, K., Hanioka, N., Masuda, K., Koeda, A., Naito, S., Miyata, A., & Narimatsu, S. (2013). Characterization of marmoset CYP2B6: CDNA cloning, protein expression and enzymatic functions. Biochemical Pharmacology, 85(8), 1182-1194. https://doi.org/10.1016/j.bcp.2013.01.024

Characterization of marmoset CYP2B6 : CDNA cloning, protein expression and enzymatic functions. / Mayumi, Kei; Hanioka, Nobumitsu; Masuda, Kazufumi; Koeda, Akiko; Naito, Shinsaku; Miyata, Atsuro; Narimatsu, Shizuo.

In: Biochemical Pharmacology, Vol. 85, No. 8, 15.04.2013, p. 1182-1194.

Research output: Contribution to journalArticle

Mayumi, K, Hanioka, N, Masuda, K, Koeda, A, Naito, S, Miyata, A & Narimatsu, S 2013, 'Characterization of marmoset CYP2B6: CDNA cloning, protein expression and enzymatic functions', Biochemical Pharmacology, vol. 85, no. 8, pp. 1182-1194. https://doi.org/10.1016/j.bcp.2013.01.024
Mayumi, Kei ; Hanioka, Nobumitsu ; Masuda, Kazufumi ; Koeda, Akiko ; Naito, Shinsaku ; Miyata, Atsuro ; Narimatsu, Shizuo. / Characterization of marmoset CYP2B6 : CDNA cloning, protein expression and enzymatic functions. In: Biochemical Pharmacology. 2013 ; Vol. 85, No. 8. pp. 1182-1194.
@article{88eec4fd1aa54a88997a791eaea01797,
title = "Characterization of marmoset CYP2B6: CDNA cloning, protein expression and enzymatic functions",
abstract = "The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2{\%} identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10{\%} that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.",
keywords = "Bupropion, cDNA cloning, Docking simulation, Efavirenz, Marmoset CYP2B6",
author = "Kei Mayumi and Nobumitsu Hanioka and Kazufumi Masuda and Akiko Koeda and Shinsaku Naito and Atsuro Miyata and Shizuo Narimatsu",
year = "2013",
month = "4",
day = "15",
doi = "10.1016/j.bcp.2013.01.024",
language = "English",
volume = "85",
pages = "1182--1194",
journal = "Biochemical Pharmacology",
issn = "0006-2952",
publisher = "Elsevier Inc.",
number = "8",

}

TY - JOUR

T1 - Characterization of marmoset CYP2B6

T2 - CDNA cloning, protein expression and enzymatic functions

AU - Mayumi, Kei

AU - Hanioka, Nobumitsu

AU - Masuda, Kazufumi

AU - Koeda, Akiko

AU - Naito, Shinsaku

AU - Miyata, Atsuro

AU - Narimatsu, Shizuo

PY - 2013/4/15

Y1 - 2013/4/15

N2 - The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.

AB - The common marmoset is a promising species for evaluating the safety of drug candidates. To further understand the capacity for drug metabolism in marmosets, a cDNA encoding a CYP2B enzyme was cloned from the total RNA fraction of marmoset liver by 3′- and 5′-RACE methods. Nucleotide and deduced amino acid sequences showed 90.8 and 86.2% identity, respectively, with human CYP2B6. The marmoset CYP2B6 (marCYP2B6) protein was expressed in insect cells, and its enzymatic properties were compared with those of human (humCYP2B6) and cynomolgus monkey (cynCYP2B6) orthologs in liver and insect cell microsomes. Enzymatic functions were examined for the oxidation of 7-ethoxy-4-(trifluoromethyl)coumarin (7-ETC), bupropion (BUP) and efavirenz (EFV). The kinetic profiles for the oxidation of the three substrates by liver microsomal fractions were similar between humans and cynomolgus monkeys (biphasic for 7-ETC and monophasic for BUP and EFV), but that of marmosets was unique (monophasic for 7-ETC and biphasic for BUP and EFV). Recombinant enzymes, humCYP2B6 and cynCYP2B6, also yielded similar kinetic profiles for the oxidation of the three substrates, whereas marCYP2B6 showed activity only for 7-ETC hydroxylation. In silico docking simulations suggested that two amino acid residues, Val-114 and Leu-367, affect the activity of marCYP2B6. In fact, a marCYP2B6 mutant with substitutions V114I and L367V exhibited BUP hydroxylase activity that was 4-fold higher than that of humCYP2B6, while its EFV 8-hydroxylase activity was only 10% that of the human enzyme. These results indicate that the amino acids at positions 114 and 367 affect the enzymatic capacity of marmoset CYP2B6.

KW - Bupropion

KW - cDNA cloning

KW - Docking simulation

KW - Efavirenz

KW - Marmoset CYP2B6

UR - http://www.scopus.com/inward/record.url?scp=84875228375&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84875228375&partnerID=8YFLogxK

U2 - 10.1016/j.bcp.2013.01.024

DO - 10.1016/j.bcp.2013.01.024

M3 - Article

VL - 85

SP - 1182

EP - 1194

JO - Biochemical Pharmacology

JF - Biochemical Pharmacology

SN - 0006-2952

IS - 8

ER -