Random mutagenesis of human cytochrome p450 2A6 and screening with indole oxidation products.

Nakamura K, Martin MV, Guengerich FP
Arch Biochem Biophys. 2001 395 (1): 25-31

PMID: 11673862 · DOI:10.1006/abbi.2001.2569

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. 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.

Copyright 2001 Academic Press.

MeSH Terms (17)

Aryl Hydrocarbon Hydroxylases Cell Membrane Chromatography, High Pressure Liquid Coloring Agents Coumarins Cytochrome P-450 CYP2A6 Cytochrome P-450 Enzyme System Escherichia coli Humans Indigo Carmine Indoles Kinetics Mixed Function Oxygenases Mutagenesis Oxidation-Reduction Spectrophotometry Umbelliferones

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