title: Functional characterization of zebrafish cytochrome P450 1 family proteins expressed in yeast.
abstract: BACKGROUND
  Zebrafish express five cytochrome P450 1 genes: CYP1A, CYP1B1, CYP1C1, CYP1C2, inducible by aryl hydrocarbon receptor agonists, and CYP1D1, a constitutively expressed CYP1A-like gene. We examined substrate selectivity of CYP1s expressed in yeast.
  
  
  METHODS
  CYP1s were expressed in W(R) yeast, engineered to over-express P450 reductase, via pYES/DEST52 and via pYeDP60. Microsomal fractions from transformed yeast were examined for activity with fluorogenic substrates, benzo[a]pyrene and testosterone. Modeling and docking approaches were used to further evaluate sites of oxidation on benzo[a]pyrene and testosterone.
  
  
  RESULTS
  CYP1s expressed in yeast dealkylated ethoxy-, methoxy-, pentoxy- and benzoxy-resorufin (EROD, MROD, PROD, BROD). CYP1A and CYP1C2 had the highest rates of EROD activity, while PROD and BROD activities were low for all five CYP1s. The relative rates of resorufin dealkylation by CYP1C1, CYP1C2 and CYP1D1 expressed via pYeDP60 were highly similar to relative rates obtained with pYES/DEST52-expressed enzymes. CYP1C1 and CYP1C2 dealkylated substituted coumarins and ethoxy-fluorescein-ethylester, while CYP1D1 did not. The CYP1Cs and CYP1D1 co-expressed with epoxide hydrolase oxidized BaP with different rates and product profiles, and all three produced BaP-7,8,9,10-tetrol. The CYP1Cs but not CYP1D1 metabolized testosterone to 6β-OH-testosterone. However, CYP1D1 formed an unidentified testosterone metabolite better than the CYP1Cs. Testosterone and BaP docked to CYP homology models with poses consistent with differing product profiles.
  
  
  CONCLUSIONS
  Yeast-expressed zebrafish CYP1s will be useful in determining further functionality with endogenous and xenobiotic compounds.
  
  
  CONCLUSIONS
  Determining the roles of zebrafish CYP1s in physiology and toxicology depends on knowing the substrate selectivity of these enzymes.
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA. Electronic address: jstegeman@whoi.edu.
authors.forename: John J
authors.initials: JJ
authors.lastname: Stegeman
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.
authors.forename: Lars
authors.initials: L
authors.lastname: Behrendt
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Woodin
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.
authors.forename: Akira
authors.initials: A
authors.lastname: Kubota
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lemaire
authors.affiliation: Laboratoire d'Ingenierie des Systèmes Biologiques et des Procédés, CNRS UMR 5504, Campus INSA, 135 Avenue de Rangueil, F-31400 Toulouse, France; Université de Toulouse, 135 Avenue de Rangueil, F-31077 Toulouse, France; 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.
authors.forename: Denis
authors.initials: D
authors.lastname: Pompon
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA.
authors.forename: Jared V
authors.initials: JV
authors.lastname: Goldstone
authors.affiliation: Laboratoire d'Ingenierie des Systèmes Biologiques et des Procédés, CNRS UMR 5504, Campus INSA, 135 Avenue de Rangueil, F-31400 Toulouse, France; Université de Toulouse, 135 Avenue de Rangueil, F-31077 Toulouse, France; 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.
authors.forename: Philippe
authors.initials: P
authors.lastname: Urban
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism; D015027:Zebrafish / Q000378:metabolism*
chemical_list: D001564:Benzo(a)pyrene; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System
keywords: Cytochrome P450 family 1; Recombinant CYP1 proteins; Substrate selectivity; Yeast; Zebrafish