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KMID : 0606920130210060487
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Biomolecules & Therapeutics
2013 Volume.21 No. 6 p.487 ~ p.492
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Evaluation of Luminescent P450 Analysis for Directed Evolution of Human CYP4A11
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Choi Seung-Hye
Han Song-Hee
Lee Hwa-Youn
Chun Young-Jin
Kim Dong-Hak
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Abstract
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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 (kcat/Km) for lauric acid hydroxylation mainly due to an increase in Km. CP2601 and CP2616 showed much greater reductions (>75%) in the catalytic efficiency due to both a decrease in kcat and an increase in Km. 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.
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KEYWORD
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P450, CYP4A11, Luciferin, Lauric acid, GC-mass spectrometry
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