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• W1993779639 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. 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. Microsomal P450 2The abbreviations used are: P450cytochrome P450OBzlOBn; and OCH2Ph; benzyloxyHPLChigh performance liquid chromatographyAPCIatmospheric pressure chemical ionizationNOESYnuclear Overhauser effect correlation spectroscopyHMBCheteronuclear multiple bond correlationCIGARconstant time inverse-detected gradient accordion rescaled long-rangeWTwild-typeMSmass spectrometry.2The abbreviations used are: P450cytochrome P450OBzlOBn; and OCH2Ph; benzyloxyHPLChigh performance liquid chromatographyAPCIatmospheric pressure chemical ionizationNOESYnuclear Overhauser effect correlation spectroscopyHMBCheteronuclear multiple bond correlationCIGARconstant time inverse-detected gradient accordion rescaled long-rangeWTwild-typeMSmass spectrometry. enzymes (also termed “heme thiolate P450” (1Palmer G. Reedijk J. J. Biol. Chem. 1992; 267: 665-677Abstract Full Text PDF PubMed Google Scholar)) are well known for their remarkable capabilities in the catalysis of diverse oxygenation reactions (2Gotoh O. J. Biol. Chem. 1992; 267: 83-90Abstract Full Text PDF PubMed Google Scholar, 3Zhao H. Giver L. Shao Z. Affholter J.A. Arnold F.H. Nat. Biotechnol. 1998; 16: 258-261Crossref PubMed Scopus (580) Google Scholar, 4Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. Kluwer Academic/Plenum Publishers, New York2005Crossref Scopus (2) Google Scholar, 5Guengerich F.P. Chem. Res. Toxicol. 2001; 14: 611-650Crossref PubMed Scopus (1339) Google Scholar, 6Guengerich F.P. Ortiz de Montellano P.R. Cytochrome P450: Structure, Mechanism, and Biochemistry. Kluwer Academic/Plenum Publishers, New York2005: 377-531Crossref Scopus (278) Google Scholar). 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 (7Guengerich F.P. Nat. Rev. Drug Discov. 2002; 1: 359-366Crossref PubMed Scopus (192) Google Scholar). 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 substrate is bound) that is obvious in the crystal structure, other residues in the protein may also play important roles. With the availability of various high throughput screening methods and robotic systems, random mutagenesis has become a widely used tool in protein engineering both to improve catalytic efficiency and to investigate structure-function relationships (8Arnold F.H. Georgiou G. Methods Mol. Biol. 2003; 230Google Scholar). For P450 enzymes, random mutagenesis and molecular breeding are emerging areas. Bacterial P450 102A1 has been used as a starting point to develop catalysts that can hydroxylate alkanes in a regio- and enantioselective manner (9Peters M.W. Meinhold P. Glieder A. Arnold F.H. J. Am. Chem. Soc. 2003; 125: 13442-13450Crossref PubMed Scopus (279) Google Scholar) or that have special features (10Seng Wong T. Arnold F.H. Schwaneberg U. Biotechnol. Bioeng. 2004; 85: 351-358Crossref PubMed Scopus (168) Google Scholar). This laboratory has developed P450 1A2 using several strategies to improve the catalytic efficiency toward several substrates (11Parikh A. Josephy P.D. Guengerich F.P. Biochemistry. 1999; 38: 5283-5289Crossref PubMed Scopus (110) Google Scholar, 12Yun C.-H. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 11319-11329Crossref PubMed Scopus (132) Google Scholar, 13Kim D. Guengerich F.P. Biochemistry. 2004; 43: 981-988Crossref PubMed Scopus (82) Google Scholar).Molecular breeding of P450 2A6 was done in this laboratory (14Nakamura K. Martin M.V. Guengerich F.P. Arch. Biochem. Biophys. 2001; 395: 25-31Crossref PubMed Scopus (77) Google Scholar), 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 (2Gotoh O. J. Biol. Chem. 1992; 267: 83-90Abstract Full Text PDF PubMed Google Scholar), and a staggered extension process method (15Stemmer W.P. Nature. 1994; 370: 389-391Crossref PubMed Scopus (1605) Google Scholar). The screening was based on the metabolism of indole, a substrate of P450 2A6 (16Gillam E.M.J. Aguinaldo A.M. Notley L.M. Kim D. Mundkowski R.G. Volkov A. Arnold F.H. Soucek P. DeVoss J.J. Guengerich F.P. Biochem. Biophys. Res. Commun. 1999; 265: 469-472Crossref PubMed Scopus (125) Google Scholar, 17Gillam E.M.J. Notley L.M. Cai H. DeVoss J.J. Guengerich F.P. Biochemistry. 2000; 39: 13817-13824Crossref PubMed Scopus (243) Google Scholar) that can be hydroxylated to a product (indoxyl) that dimerizes to the blue compound indigo. The activity of the selected mutant was enhanced toward indole as well as some substituted indoles. This mutant has been used successfully to produce various substituted indigoids with enhanced activities as potent kinase inhibitors (18Guengerich F.P. Sorrells J.L. Schmitt S. Krauser J.A. Aryal P. Meijer L. J. Med. Chem. 2004; 43: 3236-3241Crossref Scopus (70) Google Scholar, 19Wu Z.-L, Aryal, P. Lozach O. Meijer L. Guengerich F.P. Chem. Biodivers. 2005; 2: 51-65Crossref PubMed Scopus (39) Google Scholar).In the course of previous work on the biotransformation of 45 indole compounds catalyzed by the L240C/N297Q mutant, we found that this mutant showed limitations in using some substrates, among which were those bearing bulky groups (19Wu Z.-L, Aryal, P. Lozach O. Meijer L. Guengerich F.P. Chem. Biodivers. 2005; 2: 51-65Crossref PubMed Scopus (39) Google Scholar). In the x-ray crystal structure of the P450 2A6-coumarin complex recently determined by Johnson and co-workers (20Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (282) Google Scholar), the active site of P450 2A6 contains large aromatic residues that reduce the volume of the substrate-binding site. The active site is ∼6-fold smaller than that of P450 2C8 (Protein Data Bank code 1PQ2) (21Schoch G.A. Yano J.K. Wester M.R. Griffin K.J. Stout C.D. Johnson E.F. J. Biol. Chem. 2004; 279: 9497-9503Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), consistent with the size of typical substrates such as coumarin (22Yun C.-H. Shimada T. Guengerich F.P. Mol. Pharmacol. 1991; 40: 679-685PubMed Google Scholar), nicotine (23Yamazaki H. Inoue K. Hashimoto M. Shimada T. Arch. Toxicol. 1999; 73: 65-70Crossref PubMed Scopus (201) Google Scholar, 24Hecht S.S. Hochalter J.B. Villalta P.W. Murphy S.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12493-12497Crossref PubMed Scopus (128) Google Scholar), and indole.We are interested in expanding the substrate specificity of P450 2A6 using mutagenesis methods to augment the active pocket so that it can accommodate indoles with bulky substitution groups to increase the diversity of libraries of products generated for use as chemicals for screening of biological activity (19Wu Z.-L, Aryal, P. Lozach O. Meijer L. Guengerich F.P. Chem. Biodivers. 2005; 2: 51-65Crossref PubMed Scopus (39) Google Scholar). In this work, error-prone PCR was used to construct randomized libraries, and a colorimetric colony-based method was used in the screening of mutants. Two critical amino acid residue changes that allow oxidization of both 4- and 5-benzyloxy(OBzl) indoles to form colored products were identified. The results are interpreted in the context of the x-ray crystal structure (20Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (282) Google Scholar).EXPERIMENTAL PROCEDURESChemicals—Indole, 4-chloroindole, and 4- and 5-OBzl-indoles were purchased from Aldrich or Lancaster Synthesis (Windham, NH). All other reagents and solvents were obtained from general commercial suppliers and used without further purification.Spectroscopy—UV-visible spectra were recorded either with a Hewlett-Packard 1040A diode array detector (on-line with HPLC) or in Me2SO or H2O using a Cary 14/OLIS or Aminco DW2a/OLIS spectro-photometer for quantitative studies. Mass spectra were obtained in the Vanderbilt facility either on-line with HPLC or via direct infusion using a Finnigan TSQ 7000 triple quadrupole mass spectrometer (Thermo Electron Corp., San Jose, CA) equipped with a standard API-1 atmospheric pressure chemical ionization (APCI) source in the positive or negative ion mode. N2 was used as a sheath gas (50 p.s.i.). The vaporizer temperature was set to 500 °C, and the corona current was maintained at 5 μA. The capillary was set at 220 °C and 25 V (or -25 V in the negative mode). The tube lens voltage was set to 80 V (or to -96Vinthe negative mode). The collision-induced dissociation was set to -30Vin tandem mass spectrometry experiments. Data acquisition and spectral analysis were conducted with Finnigan ICIS software on a Digital Equipment Alpha workstation.All NMR experiments were carried out in the Vanderbilt facility at 298 K with Bruker Avance™ DRX 500- and 400-MHz instruments. Samples were dissolved in d6-Me2SO or CD3CN, and semiquantitative estimates of the amounts of indigoids were made by adding known amounts of CH3CO2H and referencing to this internal -CH3 signal (δ 1.91). Detailed structural information for four was obtained from four separate two-dimensional NMR experiments. Through-space 1H-1H proximity was observed using nuclear Overhauser effect correlation spectroscopy (NOESY) with a mixing time of 500 ms. Single C-H bond connectivity was obtained using heteronuclear multiple quantum correlation methods. Long-range C-H bond connectivity (two to three bonds) was obtained using heteronuclear multiple bond correlation (HMBC) with a 55-ms delay and 1H-13C CIGAR-HMBC with a delay for evolution of long-range couplings being modulated between 6 and 12 Hz and a low pass J-filter to suppress one-bond correlations. The NOESY and COSY spectra of compounds 6, 8, and 10 were recorded (mixing time of 600 ms for NOESY) to determine the hydroxylation positions. All NMR data were processed using XWIN-NMR™ software on an Octane workstation (Silicon Graphics, Mountain View, CA).Expression Systems—The system involved expression of human P450 2A6 in Escherichia coli (25Soucek P. Arch. Biochem. Biophys. 1999; 370: 190-200Crossref PubMed Scopus (53) Google Scholar). The P450 2A6 double mutant L240C/N297Q used to construct the library had been selected from libraries generated in selected regions of the P450 2A6 sequence on the basis of their ability to produce colored indigoids (14Nakamura K. Martin M.V. Guengerich F.P. Arch. Biochem. Biophys. 2001; 395: 25-31Crossref PubMed Scopus (77) Google Scholar). All of the wild-type (WT) and mutant P450 2A6 enzymes and human NADPH-cytochrome P450 reductase were expressed using a bicistronic pCW′ vector (pCW2A6bc) (26Parikh A. Gillam E.M.J. Guengerich F.P. Nat. Biotechnol. 1997; 15: 784-788Crossref PubMed Scopus (279) Google Scholar) and a tryptophanase-negative strain (trnA-)of E. coli as the host bacteria (19Wu Z.-L, Aryal, P. Lozach O. Meijer L. Guengerich F.P. Chem. Biodivers. 2005; 2: 51-65Crossref PubMed Scopus (39) Google Scholar).The expression medium contained 48 g of Terrific Broth/liter (BD Biosciences), 2.0 g of Bacto-peptone/liter (BD Biosciences), 0.4% (v/v) glycerol, 1.0 mm 5-aminolevulinic acid, 100 μg of ampicillin/liter, 1.0 mm thiamin, 1.0 mm isopropyl β-d-thiogalactopyranoside, a 0.025% (v/v) mixture of trace elements (27Sandhu P. Baba T. Guengerich F.P. Arch. Biochem. Biophys. 1993; 306: 443-450Crossref PubMed Scopus (133) Google Scholar), and 1% (v/v) starter cultures that had been grown with each newly transformed E. coli cell in LB medium (BD Biosciences) containing 100 μg of ampicillin/liter for 16 h at 32 °C with gyratory shaking at 200 rpm. The solid medium used in library screening contained 40 g of LB agar/liter (BD Biosciences), 1.0 mm 5-aminolevulinic acid, 100 μg of ampicillin/liter, 1.0 mm thiamin, 1.0 mm isopropyl β-d-thiogalactopyranoside, a 0.025% (v/v) mixture of trace elements (27Sandhu P. Baba T. Guengerich F.P. Arch. Biochem. Biophys. 1993; 306: 443-450Crossref PubMed Scopus (133) Google Scholar), and 0.5 mm 5-OBzl-indole and was cast in large bioassay Q tray plates (25 × 25 cm; Genetix, Hampshire, UK).Construction of P450 2A6 Libraries—The open reading frame region of the P450 2A6 gene (1.5 kb) was mutated using a low fidelity PCR method and amplified in a 50-μl PCR mixture containing 250 ng of pCW2A6bc-L240C/N297Q or pCW2A6bc-WT plasmid, 120 ng of forward primer 5′-TAGGAGGTCATATGGCTGCT-3′ and reverse primer 5′-ATTTCTAGACCGGAAGGCTT-3′, 2.5 units of Mutazyme ® DNA polymerase (Stratagene, La Jolla, CA), 0.2 mm each dNTP, and 10× Mutazyme® reaction buffer (Stratagene). The 1.5-kb amplified PCR library fragment was purified by agarose gel electro-phoresis and cloned into the pCW2A6bc vector using the NdeI and XbaI restriction sites. The ligation mixture was transformed into E. coli DH10B ultracompetent cells (Invitrogen) by electroporation. The library plasmid DNA was purified using a QIAprep® miniprep kit (Qiagen Inc.).Phenotypic Selection Based on Color Formation—The bicistronic library DNA was transformed into the E. coli trnA- strain by heat shock at 41 °C, plated on large bioassay Q tray plates, and allowed to grow at 30 °C for 2 days. Individual colonies with blue color were picked and grown in 3 ml of LB medium containing ampicillin (100 μg/ml) as starter cultures for 15 h at 32 °C (see Fig. 1). Plasmid DNA was prepared from the cultures using a QIAprep ® miniprep kit. 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. The plates were then incubated at 30 °C for 30 h with gyratory shaking at 250 rpm. Clones able to transform 5-OBzl-indole to form blue cultures were selected.Nucleotide Sequence Analysis—Sequencing of the plasmid DNA in the clones that produced blue cultures (following verification) was performed in the Vanderbilt facility using an Applied Biosystems Model 3700 fluorescence sequencing unit with a Taq dye terminator kit (Applied Biosystems, Foster City, CA). The mutated sequences were verified by comparing the sequences of the sense and antisense strands (entire open reading frame).Construction of Site-directed Mutants—Site-directed mutagenesis was conducted using the QuikChange ® mutagenesis kit (Stratagene) following the supplier's protocol. The primers were designed so that the mutation position was close to the middle with ∼10-15 bases of template complementary sequence on both sides and with a melting temperature of ≥75 °C (supplemental data, Table 1S). For practical purpose, a pBluescript SK(+) vector of the selected P450 2A6 mutant DNA (Bn1; ∼4.5 kb) was first constructed and subjected to site-directed mutagenesis (see Fig. 1). The 1.5-kb mutated fragment was subcloned into the pCW2A6bc vector (∼8.5 kb) using the NdeI and XbaI restriction sites. The ligation mixture was transformed into E. coli, and positive clones were confirmed following purification of plasmids. The constructed site-directed mutant plasmids were verified by sequencing both strands of DNA (see above).Whole Cell Assay for Oxidation of Indoles—Cells were incubated at 28 °C for 14 h with gyratory shaking at 200 rpm. Cell pellets containing expressed mutant P450 2A6 and NADPH-cytochrome P450 reductase were harvested from 500 ml of Terrific Broth expression medium of the E. coli trnA- strain by centrifugation at 5000 × g for 20 min, washed with 200 ml of M9 minimal medium (28Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2001Google Scholar), resuspended in 100 ml of M9 minimal medium, and used directly (after measuring the P450 concentration). The reaction mixtures consisted of 200 pmol of P450 (whole cells), 100 mm potassium phosphate buffer (pH 7.4), and varying concentrations of substrate in a total volume of 1 ml. The reactions were carried out at 37 °C in 24-well plates with gyratory shaking at 280 rpm and quenched by the addition of 0.1 ml of 0.5 m aqueous NaOH after 30 min (for indole and 4-chloroindole) or after 100 min (for 4- and 5-OBzl-indoles). The formation of colored products (indigo and indirubin) was measured by recording absorbance spectra in the visible region, and the estimated concentrations were calculated based on the extinction coefficient of the major product (subsequently determined). Estimates of kcat and Km were made using nonlinear regression with GraphPad Prism software.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 trnA- strain. The pellets were resuspended in a final volume of 10 ml of 50 mm Tris acetate (pH 7.4) containing 250 mm sucrose and 0.25 mm EDTA and homogenized with a Dounce homogenizer. The homogenized membrane fractions were stored on ice until analysis.P450 enzymes were purified from cholate extracts of E. coli membranes as described previously for P450 1A2 mutants (12Yun C.-H. Miller G.P. Guengerich F.P. Biochemistry. 2000; 39: 11319-11329Crossref PubMed Scopus (132) Google Scholar). Briefly, membranes were solubilized at 4 °C in 20 mm potassium phosphate buffer (pH 7.4) containing 20% (v/v) glycerol, 1.2 mm magnesium acetate, 10 mm β-mercaptoethanol, 0.36% (w/v) Tergitol NP-10, and 0.36% (w/v) sodium cholate. The solubilized proteins (with a C-terminal His5 tag) were purified using a nickel-nitrilotriacetic acid column (Qiagen Inc.) eluted with 400 mm imidazole. The recovered proteins were dialyzed to remove imidazole and stored at -20 °C in 50 mm potassium phosphate buffer (pH 7.7) containing 1 mm EDTA and 20% (v/v) glycerol. The proteins were >95% homogeneous as judged by SDS-PAGE; the apparent specific contents of WT P450 2A6 and the L240C/N297Q, Bn1, Bn1/1, and Bn1/4 mutants were 10.5, 10.3, 8.9, 11.2, and 9.6 nmol of P450/mg of protein, respectively, based on protein estimations done with a commercial BCA method (Pierce) used according to the manufacturer's instructions.Estimation of Substrate Binding Affinity—The binding affinity of P450 2A6 (WT and mutants) for indole or 5-OBzl-indole was estimated by titration (29Schenkman J.B. Remmer H. Estabrook R.W. Mol. Pharmacol. 1967; 3: 113-123PubMed Google Scholar). 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). The absorbance changes at 390 and 420 nm were used to estimate a spectral Kd (Ks) using GraphPad Prism software, with fitting to a one-site binding hyperbola equation using nonlinear regression.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. The reactions were carried out at 37 °C and quenched with 0.1 ml of 2 m aqueous HCl after 5 min. The product was extracted as described (30Guengerich F.P. Principles and Methods of Toxicology. Taylor & Francis, Philadelphia2001: 1625-1687Google Scholar) and assayed fluorometrically in a plate reader.Large Scale Biotransformation and Product Separation—The bio-transformations were performed in 2.8-liter Fernbach flasks with 500 ml of Terrific Broth expression medium. The cultures were incubated for 5 h at 30 °C with gyratory shaking at 250 rpm. At that time, the substrate (4- or 5-OBzl-indole) in Me2SO solution (1 m stocks) was added to the medium (0.5 ml), and shaking was continued for 22 h at 30 °C.The pelleted cells and supernatants were separated by centrifugation at 5000 × g for 30 min. The supernatant was extracted with ethyl acetate (3 × 200 ml) and dried with Na2SO4. The pellet (from 500 ml of bacterial culture) was stirred with 500 ml of (CH3)2CO and filtered through Celite® (18Guengerich F.P. Sorrells J.L. Schmitt S. Krauser J.A. Aryal P. Meijer L. J. Med. Chem. 2004; 43: 3236-3241Crossref Scopus (70) Google Scholar, 19Wu Z.-L, Aryal, P. Lozach O. Meijer L. Guengerich F.P. Chem. Biodivers. 2005; 2: 51-65Crossref PubMed Scopus (39) Google Scholar). The ethyl acetate and (CH3)2CO extracts were combined and concentrated in vacuo, and the resulting solids were fractionated by semipreparative HPLC. HPLC was done on a Beckman Ultra[-sphere column (10 × 250 mm, 5 μm) with a CH3CN/H2O mobile phase at a flow rate of 3 ml/min with 40% (v/v) CH3CN (in H2O) for 10 min, a 40-60% CH3CN linear gradient over 20 min, a 60-80% CH3CN linear gradient over 10 min, and a 80-100% CH3CN linear gradient over 5 min. To separate compounds 5 and 6, HPLC conditions of 30% (v/v) CH3CN (in H2O) for 10 min and a 30-35% CH3CN linear gradient over 20 min were used with the same column. Detection was at 254 or 600 nm (single wavelength) or with the on-line HPLC/diode array detector system. Peaks were collected manually, and CH3CN was removed with a rotary evaporator. The remaining aqueous phase was lyophilized to dryness. In analytical systems, a Beckman Ultrasphere HPLC column (4.6 × 250 mm, 5 μm) was used with a CH3CN/H2O or CH3OH/H2O mobile phase at a flow rate of 1 ml/min.Substrate Docking and Modeling of the P450 2A6 N297Q/I300V Mutant—The coordinates of the P450 2A6 N297Q/I300V mutant were built by introducing substitutions into the WT P450 2A6 coordinates (20Yano J.K. Hsu M.H. Griffin K.J. Stout C.D. Johnson E.F. Nat. Struct. Mol. Biol. 2005; 12: 822-823Crossref PubMed Scopus (282) Google Scholar) in silico using interactive graphics techniques in the program O (31Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar), followed by manual docking of each of the two substrates into the binding site cavity. A rotamer of Gln capable of donating a hydrogen bond to the substrate bound in the active site was selected. Subsequent energy minimization (conjugate gradient method) was performed using a CNS software routine with no experimental energy term (32Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar). Energy minimization resulted in molecular models of P450 2A6 with bound substrates having a root mean square deviation of 0.55 Å for C-α atoms compared with the original structure. Subsequently, ligand coordinates were removed, and protein coordinates were used as a rigid receptor for docking of these same substrates using the FlexX Suite module of Tripos SYBYL software (33Rarey M. Kramer B. Lengauer T. Klebe G. J. Mol. Biol. 1996; 261: 470-489Crossref PubMed Scopus (2339) Google Scholar), including the pharmacophore constraint of Gln297 in the enzyme active site as a hydrogen bond donor.RESULTSLibrary Screening—Two randomized libraries were constructed using error-prone PCR following the manufacturer's instructions (Stratagene) to adjust the mutation rate to zero to three mutations/1000 bp using either WT P450 2A6 or the double mutant L240C/N297Q as a template (Fig. 1). The screening method was a convenient colorimetric colony-based method that allows rapid screening of thousands of colonies by simply plating them on LB expression plates fortified with the target substrate (5-OBzl-indole in this case) and picking up the blue colonies directly from the plate. LB expression medium was used instead of Terrific Broth to decrease the background color. A tryptophanase-negative strain (trnA-) of E. coli (19Wu Z.-L, Aryal, P. Lozach O. Meijer L. Guengerich F.P. Chem. Biodivers. 2005; 2: 51-65Crossref PubMed Scopus (39) Google Scholar) was used to avoid the contribution of indole to the products, i.e. to force the P450 2A6 mutants to use only OBzl-substituted indoles. Because of the temperature sensitivity of this strain (λ prophage can be switched on at 42 °C) (34Yu D. Ellis H.M. Lee E.C. Jenkins N.A. Copeland N.G. Court D.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5978-5983Crossref PubMed Scopus (1354) Google Scholar), a slightly lower temperature than 42 °C was used for the heat shock to achieve a sufficient number of colonies to work with. A total number of ∼3 × 103 colonies were screened from each of the two libraries. No blue colonies were found upon screening the WT P450 2A6-derived library. The double mutant-derived library yielded three colonies, designated Bn1 (with a clear blue color), Bn2, and Bn3 (with a brown color), among which only Bn1 yielded a distinctive blue color when incubated in 1 ml of Terrific Broth expression medium fortified with 5-OBzl-indole (Fig. 1). This mutant was determined to be P450 2A6 I140M/L240C/N297Q/I300V/I366V by sequence analysis, with three more mutation positions added to the template DNA.Critical Residue Identification and Kinetic Parameters for WT P450 2A6 and Mutants—To determine which mutation positions were critical in gaining the ability to oxidize 5-OBzl-indole, 13 site-directed mutants were constructed from the Bn1 mutant, with one to four mutation sites recovered (Fig. 1 and" @default.
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• W1993779639 date "2005-12-01" @default.
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• W1993779639 title "Expansion of Substrate Specificity of Cytochrome P450 2A6 by Random and Site-directed Mutagenesis*" @default.
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