Abstract
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-6'H-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.
Highlights
Diverse oxygenation reactions [2,3,4,5,6]
Molecular breeding of P450 2A6 was done in this laboratory [14], and a double mutant (L240C/N297Q) was selected from libraries constructed by random mutagenesis using randomized primers, each covering four positions of one of the substrate recognition site regions [2], and a staggered extension process method [15]
The screening was based on the metabolism of indole, a substrate of P450 2A6 [16, 17] that can be hydroxylated to a product that dimerizes to the blue compound indigo
Summary
Chemicals—Indole, 4-chloroindole, and 4- and 5-OBzl-indoles were purchased from Aldrich or Lancaster Synthesis (Windham, NH). Verification of the new mutants was performed by inoculating the starter cultures into 1 ml of Terrific Broth expression medium (1%, v/v) fortified with 1 mM 5-OBzl-indole on 24-well plates. Preparation of Membranes and Purification of Recombinant WT P450 2A6 and Mutants—Bacterial inner membranes containing mutant P450 2A6 and NADPH-cytochrome P450 reductase were isolated from 500 ml of Terrific Broth expression medium of the E. coli trnAϪ strain. Substrates were dissolved in Me2SO and added to 1.0 ml of 100 mM potassium phosphate buffer (pH 7.4) containing 2– 4 nmol of purified enzyme, and spectra were recorded following each addition (350 –500 nm). Coumarin 7-Hydroxylation Assay—The reaction mixtures contained 20 pmol of P450 (cell membranes), 100 mM potassium phosphate buffer (pH 7.4), and varying concentrations of coumarin (from a 100-fold stock in H2O) in a total volume of 0.5 ml. A Beckman Ultrasphere HPLC column (4.6 ϫ 250 mm, 5 m) was used with a CH3CN/H2O or CH3OH/H2O mobile phase at a flow rate of 1 ml/min
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