title: Heterologous expression of CYP102A5 variant from Bacillus cereus CYPPB-1: Validation of model for predicting drug metabolism of human P450 probe substrates.
abstract: CYP102A5 variant (ADL27534) from isolated Bacillus cereus CYPPB-1 was heterologously expressed in Escherichia coli Top 10 cells. Comparative sequence analysis of purified CYP102A5 variant with respect to reported CYP102A5 (AAP10153) from Bacillus cereus ATCC 14579 revealed amino acid sequence changes at positions P245S and M318I of heme domain. The binding affinities of 15 selected human P450 probe substrates towards isolated CYP102A5 were analyzed in silico using a homology model together with molecular docking techniques to predict the human drug metabolism. In vitro analysis suggested that the purified CYP102A5 metabolizes typical substrates of human CYP2C9, CYP2D6, CYP2E1, and CYP3A4, such as coumarin, propranolol, aniline, chlorzoxazone, p-nitrophenol, and nifedipine. The calculated K M values for propranolol, chloroxazone, coumarin, aniline, and 4-nitrophenol were calculated to be 0.962 ± 0.041, 1.254 ± 0.057, 2.859 ± 0.083, 2.732 ± 0.106, and 2.528 ± 0.11 mM, respectively. Importantly, taking a ChemScore cutoff value of -31 kJ/mol, substrate binding at active site and in vitro activity as the distinguishing lines between "substrates" and "nonsubstrates" revealed one false-positive and one false-negative results out of the 15 compounds examined. This is the first report on validation of CYP102A family homology model for in silico prediction of human drug metabolism.
authors.affiliation: Department of Bioengineering and Environmental Centre, Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500 607, India.
authors.forename: Brahmaiah
authors.initials: B
authors.lastname: Pendyala
authors.forename: Subba Rao
authors.initials: SR
authors.lastname: Chaganti
authors.forename: Sathish
authors.initials: S
authors.lastname: Thadikamala
authors.forename: Prakasham
authors.initials: P
authors.lastname: Reddy Shetty
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001409:Bacillus cereus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008658:Inactivation, Metabolic; D007700:Kinetics; D062105:Molecular Docking Simulation; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases