[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and >10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.
Biomolecules and Therapeutics 11/2013; 21(6):487-492. · 0.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F-G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased K(m) values whereas CYP2A6*18 and *19 showed decreased k(cat) values, which resulted in lower catalytic efficiencies (k(cat)/K(m)). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased K(m) value, whereas CYP2A6*18 and *35 showed much greater decreases in k(cat) values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.
[Show abstract][Hide abstract] ABSTRACT: The metabolic activation of many carcinogenic heterocyclic and aryl amines are attributed to hepatic cytochrome P450 enzymes including P450 1A2. Bioactivation mechanism analysis of P450 1A2 in the limited crystallographic data requires the generation of a large number of mutants. In this study, the SRS2 region in P450 1A2 was randomly mutated with random primers designed for the non-wild type using whole-plasmid random mutagenesis, followed the screening of the mutant library. Eight mutants in Asn222 and Glu225 were selected after Escherichia
coli genotoxicity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). Each mutant E. coli membrane was used to determine k
cat and K
m values for phenacetin and 7-ethoxyresorufin O-deethylation. Most mutants showed considerably increased activity for phenacetin. The Glu225Asn and Asn222Lys mutants increased catalytic efficiencies (k
m) by 5- and 4-fold, respectively. However, the catalytic efficiencies of 7-ethoxyresorufin O-deethylation were not increased mainly because of the increase of K
m values. The Glu225Asn and Asn222Lys mutants showed increased k
cat values but exhibited greater increases in K
m values, which resulted in lower catalytic efficiencies (k
m) for 7-ethoxyresorufin O-deethylation. These results suggested that structural changes of metabolic enzymes in the process of mutagenesis result in different effects of activity by its substrates.
Molecular and Cellular Toxicology 9(1). · 0.72 Impact Factor