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 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.