Identification of amino acid residues involved in 4-chloroindole 3-hydroxylation by cytochrome P450 2A6 using screening of random libraries.
Cytochrome P450 (P450) 2A6 is able to catalyze indole hydroxylation to form the blue dye indigo. The wild-type 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(cat) 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.
Zhang
Zhi-Gang
ZG
Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu 610041, China.
Liu
Yan
Y
Guengerich
F Peter
FP
Matse
Johannes H
JH
Chen
Jun
J
Wu
Zhong-Liu
ZL
D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D030562:Databases, Protein; D004926:Escherichia coli; D006801:Humans; D006900:Hydroxylation; D007203:Indigo Carmine; D007211:Indoles / Q000378:metabolism*; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D016296:Mutagenesis; D020539:Sequence Analysis, Protein
D007211:Indoles; C062517:4-chloroindole; C030374:indole; D007203:Indigo Carmine; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6