title: Multiple steroid-binding orientations: alteration of regiospecificity of dehydroepiandrosterone 2- and 7-hydroxylase activities of cytochrome P-450 2a-5 by mutation of residue 209.
abstract: The mutation of Ala-117 to Val conferred dehydroepiandrosterone (DHEA) hydroxylase activity on cytochrome P-450 2a-4, with the production of both 2 alpha- and 7 alpha-hydroxyDHEA at similar rates. P-450 2a-5 which has Val at position 117, acquired high DHEA hydroxylase activity by mutation of Phe-209. Mutant F209L of P-450 2a-5 exhibited strong regiospecificity at the 2-position of the DHEA molecule with the production of 2 alpha-hydroxy DHEA as the major metabolite. On the other hand, mutant F209V of P-450 2a-5 showed the 7-position to be the major hydroxylation site, 7 beta-hydroxyDHEA and 7 alpha-OHDHEA being produced. Therefore the regiospecificity of DHEA hydroxylase activity of P-450 2a-5 is altered between the 2- and 7-position depending on the amino acid at position 209. Modelling of the DHEA molecule in the pocket of bacterial P-450cam showed that the steroid can be accommodated in at least two orientations for which the 2- or 7- position is near the sixth axial position of the haem. Moreover, these two orientations, which are of similar energy, can be interconverted by a 180 degrees rotation of the steroid molecule around its long axis. These results support the hypothesis that the steroid molecule in the pocket is in dynamic equilibrium with multiple binding orientations and that the equilibrium is apparently determined by a few critical residues including those at positions 117 and 209.
authors.affiliation: National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709.
authors.forename: M
authors.initials: M
authors.lastname: Iwasaki
authors.forename: D G
authors.initials: DG
authors.lastname: Davis
authors.forename: T A
authors.initials: TA
authors.lastname: Darden
authors.forename: L G
authors.initials: LG
authors.lastname: Pedersen
authors.forename: M
authors.initials: M
authors.lastname: Negishi
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003687:Dehydroepiandrosterone / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D006418:Heme; D003687:Dehydroepiandrosterone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6