Characterization of marmoset CYP2B6: cDNA cloning, protein expression and enzymatic functions
| Autor: | Akiko Koeda, Shizuo Narimatsu, Shinsaku Naito, Atsuro Miyata, Kei Mayumi, Nobumitsu Hanioka, Kazufumi Masuda |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Cyclopropanes
Insecta Molecular Sequence Data Hydroxylation Biochemistry Structure-Activity Relationship chemistry.chemical_compound biology.animal Complementary DNA Animals Humans Amino Acid Sequence Cloning Molecular Bupropion Peptide sequence Pharmacology chemistry.chemical_classification Base Sequence biology Marmoset Callithrix Oxidoreductases N-Demethylating Molecular biology Benzoxazines Amino acid Cytochrome P-450 CYP2B6 Macaca fascicularis Enzyme chemistry Alkynes Microsomes Liver Microsome Aryl Hydrocarbon Hydroxylases Drug metabolism |
| Zdroj: | Biochemical Pharmacology. 85:1182-1194 |
| ISSN: | 0006-2952 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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