dard curve of 7-hydroxycoumarin which was constructed PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19652084 in the range of 15.632000 pmol/well previously. Current assay also included control for fluorescent interference which consisted of inhibitor and bacterial control membranes. Fluorescence interference from the control incubation was generally negligible. In experiments involving pre-incubation, the incubations were carried out as described above except that after a few minutes of prewarming, CYP2A6 was incubated with 8-MOP and NADPH generating system for 10 minutes without coumarin. Coumarin was only added after 10 minutes and incubations proceeded for a further 25 minutes. For corresponding incubations without pre-incubation, all reaction components were added from the beginning and incubated for a total time of 35 minutes. All reactions were incubated in triplicate with replications in separate experiments. Due to photosensitivity of 8-MOP, the assay was conducted using 96-well plates with black polystyrene wells that came with lid. Furthermore, all the 8-MOP stocks were prepared and stored in amber glass tubes or tubes sealed or covered with proper covering to protect it from light. Establishment of CYP2A6 Monooxygenase Systems in E. coli A fully functional P450 monooxygenase system requires collaboration of the CYP of interest in concert with the essential coenzyme, NADPH-CYP oxidoreductase. Here, we co-expressed the individual CYP2A6 plasmid constructs pCWCYP2A6, pCW-CYP2A615, pCW-CYP2A616, pCWCYP2A621 or pCW-CYP2A622, with pACYC-OxR plasmid which allowed for OxR co-expression that was important for CYP2A6 catalytic function. Preparation of membrane fragments expressing holoenzyme protein of respective CYP2A6 construct was in accordance to the established method described previously. Membrane fractions were stored at 280uC in a 1:1 mixture of pH 7.6 TES and ice-cold distilled water before use. Kinetic Analysis Enzyme kinetic data were analyzed by nonlinear least squares regression 
analysis software EZ-FitTM of which the kinetic parameters, Michaelis-Menten constant was determined over the substrate range UNC0642 biological activity studied. The IC50 values for the 8-MOP inhibition studies were interpolated mathematically by SigmaPlotH software. All experimental data were expressed as means and standard deviations. Comparison of the means was analysed using Student’s t-test and one way ANOVA with SPSS. Molecular Docking and Virtual Mutation of CYP2A6 All computational works were performed on Discovery Studio 3.5. The X-ray crystal structure of CYP2A6 was retrieved from the Protein Data Bank . The same binding site was used for all docking works involving 8-MOP. 8-MOP was constructed using ChemBIoDraw Ultra 2012. The file was opened in DS and energy minimization was carried out by CHARMm force field. The optimization protocol was applied in preparation of the protein and docking procedures. The CDOCKER program was subsequently used for the docking. The CDOCKER is CHARMm-based docking algorithm that uses the CHARMm family of force fields and algorithm adopts a strategy involving generation of several initial ligand orientations in the active site of target protein followed by molecular dynamics -based CYP2A6 Inactivation Assay Assessment of coumarin 7-hydroxylation is a signature marker for CYP2A6 metabolic assay. To evaluate the inhibitory potency of 8-MOP on the kinetic activity of CYP2A6 variants, we adopted a fluorescence-based coumarin 7-hydroxylase assay upon minor modifications of an established protoc
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