Analysis of Coumarin 7-Hydroxylation Activity of Cytochrome P450 2A6 using Random Mutagenesis

Analysis of coumarin 7-hydroxylation activity of cytochrome P450 2A6 using random mutagenesis.

Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k(cat) and Km) for ...

Inter-individual Variability of Coumarin 7-hydroxylation (CYP2A6 activity) in an Iranian Population

Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes.

Three semi-rational approaches, combinatorial site-saturation mutagenesis (CSSM) using a reduced amino acid set and two libraries based on C(orbit) and CRAM computational design algorithms targeting up to 10 active site residues, were used to engineer cytochrome P450 BM3 to demethylate dimethyl ether and hydroxylate propane and ethane. These small libraries (343-1028 variants) were all enriched...

Induction of Cytochrome P450 2A6 by Bilirubin in Human Hepatocytes

The influence of bilirubin on mRNA expression of cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT) and nuclear receptors in human hepatocytes was investigated. The treatment of the hepatocytes with 40 μg/mL bilirubin, which corresponds to hyperbilirubinemia, resulted in 1.7-fold increase of CYP2A6 mRNA compared to the vehicle control while CYP2A6 mRNA did not change after treatment with ...

2'-Hydroxylation of nicotine by cytochrome P450 2A6 and human liver microsomes: formation of a lung carcinogen precursor.

Smokers or people undergoing nicotine replacement therapy excrete approximately 10% of the nicotine dose as 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) and 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid). Previously, these acids were thought to arise by secondary metabolism of the major nicotine metabolite cotinine, but our data did not support this mechanism. Therefore, we hypothesized th...