title: Role of a mutated residue at the entrance of the substrate access channel in cytochrome p450 engineered for vitamin D(3) hydroxylation activity.
abstract: The cytochrome P450 enzyme engineered for enhancement of vitamin D(3) (VD(3)) hydroxylation activity, Vdh-K1, includes four mutations (T70R, V156L, E216M, and E384R) compared to the wild-type enzyme. Plausible roles for V156L, E216M, and E384R have been suggested by crystal structure analysis (Protein Data Bank  3A50 ), but the role of T70R, which is located at the entrance of the substrate access channel, remained unclear. In this study, the role of the T70R mutation was investigated by using computational approaches. Molecular dynamics (MD) simulations and steered molecular dynamics (SMD) simulations were performed, and differences between R70 and T70 were compared in terms of structural change, binding free energy change (PMF), and interaction force between the enzyme and substrate. MD simulations revealed that R70 forms a salt bridge with D42 and the salt bridge affects the locations and the conformations of VD(3) in the bound state. SMD simulations revealed that the salt bridge tends to be formed strongly when VD(3) passes through the binding pocket. PMFs showed that the T70R mutation leads to energetic stabilization of enzyme-VD(3) binding in the region near the heme active site. Interestingly, these results concluded that the D42-R70 salt bridge at the entrance of the substrate access channel affects the region near the heme active site where the hydroxylation of VD(3) occurs; i.e., it is thought that the T70R mutation plays an important role in enhancing VD(3) hydroxylation activity. A significant future challenge is to compare the hydroxylation activities of R70 and T70 directly by a quantum chemical calculation, and three-dimensional coordinates of the enzyme and VD(3) obtained from MD and SMD simulations will be available for the future challenge.
authors.affiliation: Green Innovation Research Laboratories, NEC Corporation, 34, Miyukigaoka, Tsukuba, Ibaraki 305-8501, Japan. hfukunishi@bu.jp.nec.com
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Fukunishi
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Yagi
authors.forename: Ken'ichi
authors.initials: K
authors.lastname: Kamijo
authors.forename: Jiro
authors.initials: J
authors.lastname: Shimada
mesh_terms: D001665:Binding Sites; D002762:Cholecalciferol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D006900:Hydroxylation; D056004:Molecular Dynamics Simulation; D009154:Mutation; D013250:Steroid Hydroxylases / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C406322:vitamin D 1-alpha hydroxylase