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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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논문 기본 정보
- 자료유형
- 학술저널
- 저자정보
- 발행연도
- 2013.1
- 수록면
- 487 - 492 (6page)
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초록· 키워드
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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 ($k_{cat}/K_m$) 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 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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참고문헌 (19)
참고문헌 신청
Adas, F. / 1999 / Requirement for omega and (omega-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11 / J. Lipid Res 40:1990~1997
Branchini, B. R. / 1989 / Naphthyl- and quinolylluciferin: green and red light emitting fi refl y luciferin analogues / Photochem. Photobiol 49:689~695
Cali, J. J. / 2006 / Luminogenic cytochrome P450 assays / Expert Opin. Drug Metab. Toxicol 2:629~645
Gainer, J. V. / 2005 / Functional variant of CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential hypertension / Circulation 111:63~69
Guengerich, F. P. / 2005 / Human cytochrome P450 enzymes. In Cytochrome P450: Structure, Mechanism, and Biochemistry / Plenum Press :377~551
Branchini, B. R. / 1989 / Naphthyl- and quinolylluciferin: green and red light emitting fi refl y luciferin analogues / Photochem. Photobiol 49:689~695
Cali, J. J. / 2006 / Luminogenic cytochrome P450 assays / Expert Opin. Drug Metab. Toxicol 2:629~645
Gainer, J. V. / 2005 / Functional variant of CYP4A11 20-hydroxyeicosatetraenoic acid synthase is associated with essential hypertension / Circulation 111:63~69
Guengerich, F. P. / 2005 / Human cytochrome P450 enzymes. In Cytochrome P450: Structure, Mechanism, and Biochemistry / Plenum Press :377~551
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