Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k cat , 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (ϳ0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process.

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Cytochrome P450 (P450) 2A6 is an important human enzyme involved in the metabolism of many xenobiotic chemicals including coumarin, indole, nicotine, and carcinogenic nitrosamines. A combination of random mutagenesis and high-throughput screening was used in the analysis of P450 2A6, utilizing a fluorescent coumarin 7-hydroxylation assay. The steady-state kinetic parameters (k cat and K m ) for coumarin 7-hydroxylation by wild-type P450 2A6 and 35 selected mutants were measured and indicated that mutants throughout the coding region can have effects on activity. Five mutants showing decreased catalytic efficiency (k cat /K m ) were further analyzed for substrate selectivity and binding affinities and showed reduced catalytic activities for 7-methoxycoumarin O-demethylation, tert-butyl methyl ether O-demethylation, and indole 3-hydroxylation. All mutants except one (K476E) showed decreased coumarin binding affinities (and also higher K m values), indicating that this is a major basis for the decreased enzymatic activities. A recent x-ray crystal structure of P450 2A6 bound to coumarin (Yano, J. K., Hsu, M. H., Griffin, K. J., Stout, C. D., and Johnson, E. F. (2005) Nat. Struct. Mol. Biol. 12, 822-823) indicates that the recovered A481T and N297S mutations appear to be close to coumarin, suggesting direct perturbation of substrate interaction. The decreased enzymatic activity of the K476E mutant was associated with decreases both in NADPH oxidation and the reduction rate of the ferric P450 2A6-coumarin complex. The attenuation is caused in part to lower binding affinity for NADPH-P450 reductase, but the K476E mutant did not achieve the wild-type coumarin 7-hydroxylation activity even at high reductase concentrations. Cytochrome P4502 enzymes are the major catalysts involved in the oxidative metabolism of xenobiotic chemicals, a significant focus in the areas of toxicology, drug metabolism, and pharmacology (3-5). Human P450 2A6 was identified as the major coumarin 7-hydroxlylase in humans (6 -8), and this enzyme also plays an important role in the metabolism of many xenobiotics including coumarin, nicotine, and tobacco-specific nitrosamines (8 -12). Genetic polymorphisms have been identified, and their relevance to cancer risk has been proposed because of the variations in nicotine and N-nitrosamine metabolism (13-17). The oxidation of indoles by P450 2A6 has been characterized, and the new kinase inhibitors have been biosynthesized using P450 2A6 mutants (18 -22).Recently x-ray crystal structures have been reported for P450 2A6 bound with coumarin and methoxsalen (23). The active site of P450 2A6 is six times smaller than that of human P450 2C8 (23, 24), even smaller than that of bacterial P450 101A1 (25). The decrease in size of the active site in P450 2A6 relative to P450 2C8 is caused by repositioning of helix BЈ and helices F to H toward the active site. Large aromatic residues in the active site of P450 2A6 also reduce the volume of the substrate binding site.In recent years, random mutagenesis and high-thro...

The natural product indole is a substrate for cytochrome P450 2A6. Mutagenesis of P450 2A6 was done to expand its capability in the oxidization of bulky substituted indole compounds, which are not substrates for the wild-type enzyme or the double mutant L240C/N297Q, as determined in our previous work (Wu, Z.-L., Aryal, P., Lozach, O., Meijer, L., and Guengerich, F. P. (2005) Chem. Biodivers. 2, 51-65). Error-prone PCR and site-directed mutagenesis led to the identification of two critical amino acid residue changes (N297Q and I300V) that achieve the purpose. The new mutant (N297Q/I300V) was able to oxidize both 4-and 5-benzyloxy(OBzl)indoles to form colored products. Both changes were required for oxidation of these bulky substrates. The colored product derived from 5-OBzl-indole was mainly 5,5-di-OBzl-indirubin, whereas the dominant blue dye isolated upon incubations with 4-OBzl-indole was neither an indigo nor an indirubin. Two-dimensional NMR experiments led to assignment of the structure as 4-OBzl-2-(4-OBzl-1,7-dihydro-7-oxo-6H-indol-6-ylidene)indolin-3-one, in which a pyrrole ring and a benzene ring are connected with a double bond instead of the pyrrole-pyrrole connection of other indigoids. Monomeric oxidation products were also isolated and characterized; three phenols (4-OBzl-1H-indol-5-ol, 4-OBzl-1H-indol-6-ol, and 4-OBzl-1H-indol-7-ol) and one quinone (4-OBzl-1H-indole-6,7-dione, the postulated immediate precursor of the final blue dye) were identified. The results are interpreted in the context of a crystal structure of a P450 2A6-coumarin complex. The I300V change opens an additional pocket to accommodate the OBzl bulk. The N2297Q change is postulated to generate a hydrogen bond between Gln and the substrate oxygen. Thus, the substrate specificity of P450 2A6 was expanded, and new products were obtained in this study. Microsomal P4502 enzymes (also termed "heme thiolate P450" (1)) are well known for their remarkable capabilities in the catalysis of diverse oxygenation reactions (2-6). These enzymes have been studied mostly as the principal catalysts involved in sterol synthesis, drug metabolism, and xenobiotic disposition. The use of P450 enzymes in the area of biocatalysis and fine chemical production is still largely unexploited (7). Random mutagenesis is one of the main approaches in terms of developing P450 enzymes with new functions and can also be used to enhance the knowledge of structure-function relationships of these enzymes and thus provide more information for drug development.Random mutagenesis and molecular breeding approaches can be very useful when there is no crystal structure available to perform rational design, which is still the case for most P450 enzymes today. Furthermore, even rational designs based on crystal structures do have limitations because the enzymes possess both rigidity and flexibility. Predictions are even more complex in the case of oxidoreductase-catalyzed reactions when several factors are involved in the catalytic cycle. In addition to the active site (where ...