Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products.
Cytochrome P450 (P450) 2A6 mutants from randomized libraries generated in the substrate recognition sequence (SRS) regions were screened in Escherichia coli on the basis of indole metabolism. SRS 3 and 4 libraries yielded colonies that produced indigo at least as well as wild-type (WT) P450 2A6, and some colonies were consistently more blue upon replating. One mutant, F209T, showed indole 3-hydroxylation <WT but had a k(cat) for coumarin 7-hydroxylation 13-fold >WT. The double mutant L240C/N297Q consistently produced very blue colonies. Five mutants yielded mixtures of pigments from indole different than WT, as judged by visible spectra and HPLC of products. When bacteria expressing the mutants were grown in the presence of each of 26 substituted indoles, a variety of patterns of formation of different dyes was seen with several of the mutants. This approach has potential value in understanding P450 2A6 function and generating new dyestuffs and other products.
Nakamura
K
K
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146, USA.
Martin
M V
MV
Guengerich
F P
FP
D001189:Aryl Hydrocarbon Hydroxylases; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents / Q000032:analysis / Q000378:metabolism; D003374:Coumarins / Q000737:chemistry / Q000378:metabolism; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007203:Indigo Carmine; D007211:Indoles / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D014468:Umbelliferones / Q000096:biosynthesis
D004396:Coloring Agents; D003374:Coumarins; D007211:Indoles; D014468:Umbelliferones; C031477:7-hydroxycoumarin; C030374:indole; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D007203:Indigo Carmine; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6