title: Ordered chimerogenesis applied to CYP2B P450 enzymes.
abstract: BACKGROUND
  Structural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11.
  
  
  METHODS
  A series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast.
  
  
  RESULTS
  Most of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F'/G' cassette role and substrate access channels.
  
  
  CONCLUSIONS
  Some sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity.
  
  
  CONCLUSIONS
  The result provides a new basis for determining the structural elements that control functions in complex enzymes.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lautier
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Urban
authors.affiliation: Université Pierre-et-Marie-Curie, 4 Place Jussieu, 75005 Paris, France; INSERM U773, Faculté de Médecine Bichat-Beaujon, 75018 Paris, France.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Loeper
authors.affiliation: Solvay, Product Compliance, 52 rue de la Haie-Coq, 93306 Aubervilliers, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Jezequel
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Pompon
authors.affiliation: Université de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, 135 Avenue de Rangueil, F-31400 Toulouse, France. Electronic address: gilles.truan@insa-toulouse.fr.
authors.forename: Gilles
authors.initials: G
authors.lastname: Truan
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003374:Coumarins / Q000378:metabolism; D003520:Cyclophosphamide / Q000378:metabolism; D065702:Cytochrome P-450 CYP2B6 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D004285:Dogs; D006801:Humans; D007700:Kinetics; D062105:Molecular Docking Simulation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D011993:Recombinant Fusion Proteins; C056033:7-ethoxy-4-trifluoromethylcoumarin; D003520:Cyclophosphamide; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog)
keywords: CYP2B11; CYP2B6; Channel; Chimera; Cyclophosphamide; P450