title: The role of cytochrome P450 BM3 phenylalanine-87 and threonine-268 in binding organic hydroperoxides.
abstract: BACKGROUND
  Cytochrome P450 (P450) BM3, from Bacillus megaterium, catalyzes a wide range of chemical reactions and is routinely used as a model system to study mammalian P450 reactions and structure.
  
  
  METHODS
  The metabolism of 2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone (BHTOOH) and 2-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadien-1-one (BMPOOH) was examined with P450 BM3 and with the conserved T268 and F87 residues mutated to investigate their effects on organic hydroperoxide metabolism. To determine the effects of the mutations on the active site volume and architecture, the X-ray crystal structure of the F87A/T268A P450 BM3 heme domain (BMP) was determined and compared to previous structures. To investigate the interactions of the substrates with the F87 and T268 residues, BHTOOH and BMPOOH were docked into the BMP X-ray crystal structures.
  
  
  RESULTS
  Lower metabolism of BHTOOH and BMPOOH was observed in the WT P450 BM3 and the T268A P450 BM3 mutant than in the F87A and F87A/T268A P450 BM3 mutants. Large differences were found in the F-G loop regions and active site cavity volumes for the F87A mutated structures.
  
  
  CONCLUSIONS
  Analysis of the metabolism, X-ray crystal structures, and molecular docking simulations suggests that P450 BM3 activity toward BHTOOH and BMPOOH is mediated through substrate recognition by T268 and F87, and the active site cavity volume. Based on this information, a simplified representation is presented with the relative orientation of organic hydroperoxides in the P450 BM3 active site.
  
  
  CONCLUSIONS
  The metabolism results and structural analysis of this model P450 allowed us to rationalize the structural factors that influence organic hydroperoxide metabolism.
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, 240 W. Green St., Athens, GA 30602, USA. Electronic address: audie@uga.edu.
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.affiliation: Department of Medicinal Chemistry, University of Washington, HSB H-172, Seattle, WA 98195, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Katayama
authors.affiliation: Department of Medicinal Chemistry, University of Washington, HSB H-172, Seattle, WA 98195, USA.
authors.forename: Rüdiger
authors.initials: R
authors.lastname: Kaspera
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, 240 W. Green St., Athens, GA 30602, USA.
authors.forename: Kaitlyn V
authors.initials: KV
authors.lastname: Ledwitch
authors.affiliation: Department of Biological Structure and Biochemistry, University of Washington, HSB G520, Seattle, WA 98195-7420, USA.
authors.forename: Isolde
authors.initials: I
authors.lastname: Le Trong
authors.affiliation: Department of Biological Structure and Biochemistry, University of Washington, HSB G520, Seattle, WA 98195-7420, USA; Department of Biochemistry, University of Washington, HSB J405, Seattle, WA 98195-7350, USA.
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Stenkamp
authors.affiliation: Department of Pharmaceutical Sciences, University of Colorado Denver, Anschutz Medical Campus, Box C238-P15, Aurora, CO 80045, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Thompson
authors.affiliation: Department of Medicinal Chemistry, University of Washington, HSB H-172, Seattle, WA 98195, USA.
authors.forename: Rheem A
authors.initials: RA
authors.lastname: Totah
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000235:genetics; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011809:Quinones / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011809:Quinones; D013912:Threonine; D010649:Phenylalanine; C046508:2,6-di-tert-butyl-4-hydroperoxy-4-methyl-2,5-cyclohexadienone; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: Computer modeling; Cytochrome P450 102A1; Cytochrome P450 BM3; Organic hydroperoxides; Substrate specificity/selectivity; X-ray crystallography