title: Identification, modeling and ligand affinity of early deuterostome CYP51s, and functional characterization of recombinant zebrafish sterol 14α-demethylase.
abstract: BACKGROUND
  Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes.
  
  
  METHODS
  PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS.
  
  
  RESULTS
  Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26μM for ketoconazole and 0.64μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s.
  
  
  CONCLUSIONS
  Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s.
  
  
  CONCLUSIONS
  The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; School of Public Health, Harvard University, Boston, MA 02115, USA.
authors.forename: Ann Michelle Stanley
authors.initials: AM
authors.lastname: Morrison
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
authors.forename: Jared V
authors.initials: JV
authors.lastname: Goldstone
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; Institute of Life Science, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.
authors.forename: David C
authors.initials: DC
authors.lastname: Lamb
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
authors.forename: Akira
authors.initials: A
authors.lastname: Kubota
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lemaire
authors.affiliation: Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. Electronic address: jstegeman@whoi.edu.
authors.forename: John J
authors.initials: JJ
authors.lastname: Stegeman
mesh_terms: D000818:Animals; D005260:Female; D006801:Humans; D008024:Ligands; D008297:Male; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D011994:Recombinant Proteins / Q000737:chemistry*; D058886:Sterol 14-Demethylase / Q000737:chemistry* / Q000502:physiology; D013261:Sterols / Q000096:biosynthesis; D015027:Zebrafish
chemical_list: D008024:Ligands; D011994:Recombinant Proteins; D013261:Sterols; D058886:Sterol 14-Demethylase
keywords: Azole inhibition; CYP51; Deuterostome; Sterol 14α-demethylase; Sterol biosynthesis; Zebrafish