期刊
JOURNAL OF BIOTECHNOLOGY
卷 139, 期 1, 页码 12-18出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2008.09.010
关键词
Cytochrome P450; CYP2A6; Indigo; Indole hydroxylation; Random mutagenesis; High-throughput screening
资金
- 100 Talents Program of the Chinese Academy of Sciences
- Sichuan Province Science Foundation for Young Scholars [08ZQ026-023]
- U.S. Public Health Service [R37 CA90426, P30 ES00267]
- NATIONAL CANCER INSTITUTE [R37CA090426] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES [P30ES000267] Funding Source: NIH RePORTER
Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wildtype P450 2A6 enzyme was randomly mutated throughout the whole open reading frame and screened using 4-chloroindole hydroxylation, a substituted indole selected from 30 indole compounds for enhanced color development. Mutants with up to 5-fold increases of catalytic efficiency (k(cat)/K-m) and 2-fold increases in k,, were selected after two rounds of screening. Important residues located both in (e.g., Thr305) and outside the active site (e.g., Ser224) were identified. The study utilized a better substrate for indigo assay to obtain new information on the structure-functional relationship of P450 2A6 that was not revealed by previous mutagenesis studies with this enzyme. (c) 2008 Elsevier B.V. All rights reserved.
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