Random Mutagenesis of Human Cytochrome P450 2A6 and Screening with Indole Oxidation Products1

doi:10.1006/abbi.2001.2569

Archives of Biochemistry and Biophysics

Volume 395, Issue 1, 1 November 2001, Pages 25-31

https://doi.org/10.1006/abbi.2001.2569 Get rights and content

Abstract

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.

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^☆: ¹This study was supported in part by United States Public Health Service Grants R35 CA44353, R01 CA90426, and P30 ES00267 (F.P.G.).

^☆☆: ¹This study was supported in part by United States Public Health Service Grants R35 CA44353, R01 CA90426, and P30 ES00267 (F.P.G.).

^★: Current address: Department of Pharmacy, Gunma University School of Medicine, Gunma, Japan.

^★★: We did not extend our analysis of the kinetics of pigment formation to more concentrations. Sensitivity is not the issue (Fig. 3), but the estimation of K_m values for indole 3-hydroxylation is complicated by the instability of the initial enzymatic prpoduct (3-hydroxyindole) in the presence of oxygen (17) and the lag observed in the nonenzymatic oxidation of 3-hydroxyindole to indigo (Fig. 3A). Another complexity in attempting to do steady-state kinetics of indigo formation is the varying λ_max of the product (Fig. 3B), presumably a reflection of the formation of multiple products with overlapping spectra. It is possible to estimate kinetic parameters for some of the stable initial oxidation products, e.g., 2-hydroxyindole (17) (Fig. 4), but these are not related to pigment formation.

^☆☆☆: Similar results (rates and spectra) were obtained in a set of duplicate experiments in which the reactions were stopped after 20 min by the addition of 1% sodium dodecyl sulfate (to 1%, w/v).

^★★★: We have found the color patterns produced by the mutants with some of the substituted indoles to be very reproducible, in repeated assays (P. Aryal and F. P. Guengerich, unpublished observations).

³: To whom correspondence may be addressed at Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building (Medical Research Building I), 23rd and Pierce Avenues, Nashville, TN 37232-0146. Fax: (615) 322-3141. E-mail: [email protected].

⁴: Abbreviations used: P450, cytochrome P450 [also termed heme-thiolate P450 (1)]; WT, wild-type (P450 2A6); NPR, NADPH-P450 reductase; PCR, polymerase chain reaction; SRS, substrate recognition sequence; StEP, staggered extension process; IPTG, isopropyl-β- $d$ -thiogalactoside; LB, Luria broth.

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Regular ArticleRandom Mutagenesis of Human Cytochrome P450 2A6 and Screening with Indole Oxidation Products1☆,☆☆,★,★★,☆☆☆,★★★

Abstract

Bioorg. Medicinal Chem.

Curr. Opin. Struct. Biol.

Curr. Opin. Chem. Biol.

J. Mol. Catal., BP Enzymatic

Biochim. Biophys. Acta

Metabolism

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Biol. Chem.

Endocr. Res.

Regular Article
Random Mutagenesis of Human Cytochrome P450 2A6 and Screening with Indole Oxidation Products¹☆,☆☆,★,★★,☆☆☆,★★★