Cytochorome P450 (P450) enzymes comprise a superfamily of monooxygenases that are of considerable interest as the most versatile redox enzymes known . Molecular breeding and utilization in chemical synthesis of P450s are considered two of the emerging areas today . In the case of human P450 2A6 ,a major enzyme involved in nicotine metabolism , extensive studies involving molecular breeding and novel protein kinase inhibitor synthesis have been done following the observation of blue pigment formation (indigo) from cultures that expresses bicistronic P450 2A6 . Recently , alternate methods of complementary values were used to explore the capacity of the enzymes . 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 (kcat/Km) and 2-fold increase in kcat 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 substate 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 . In another work , to further expand the substrate spectrum of P450 2A6 , two random libraries were screened , but yield no gain-of-function mutant . Site –directed mutagenesis , however , generated a new mutant P450 2A6(N297Q/1300A) with expanded substrate specificity compared with the wild type enzyme and previously reported mutants . The new mutant was able to catalyze the oxidation of 6-benzyloxyindole to form colored indigoid dimers as analyzed with mass spectrometry . Both amino acid residue changes were required for the reaction . The results are interpreted in the context of the recently resolved crystal structures of P450 2A6 mutants . (Supported by 100 Talents Progrme of the Chinese Academy of Sciences and Sichuan Province Science Foundation for Young Scolars , 2008-23-363) .