Ann Michelle Stanley Morrison1, Jared V Goldstone2, David C Lamb3, Akira Kubota2, Benjamin Lemaire2, John J Stegeman4. 1. Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA; School of Public Health, Harvard University, Boston, MA 02115, USA. 2. Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. 3. 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. 4. Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA. Electronic address: jstegeman@whoi.edu.
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. GENERAL SIGNIFICANCE: The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.
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-mammaliandeuterostomes. METHODS: PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafishDanio rerio, the reef fish sergeant majorAbudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafishCYP51 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. purpuratusCYP51 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 zebrafishCYP51 gave KS (dissociation constant) values of 0.26μM for ketoconazole and 0.64μM for propiconazole. Displacement of carbon monoxide also indicated zebrafishCYP51 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. GENERAL SIGNIFICANCE: The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications.
Authors: Fredrik Ronquist; Maxim Teslenko; Paul van der Mark; Daniel L Ayres; Aaron Darling; Sebastian Höhna; Bret Larget; Liang Liu; Marc A Suchard; John P Huelsenbeck Journal: Syst Biol Date: 2012-02-22 Impact factor: 15.683
Authors: Jared V Goldstone; Munirathinam Sundaramoorthy; Bin Zhao; Michael R Waterman; John J Stegeman; David C Lamb Journal: Mol Phylogenet Evol Date: 2015-10-03 Impact factor: 4.286
Authors: Benjamin Lemaire; John J Stegeman; Akira Kubota; Conor M O'Meara; David C Lamb; Robert L Tanguay; Jared V Goldstone Journal: Toxicol Appl Pharmacol Date: 2016-02-04 Impact factor: 4.219
Authors: Andrew G S Warrilow; Claire M Hull; Nicola J Rolley; Josie E Parker; W David Nes; Stephen N Smith; Diane E Kelly; Steven L Kelly Journal: Appl Environ Microbiol Date: 2014-08-01 Impact factor: 4.792
Authors: Kirill V Pankov; Andrew G McArthur; David A Gold; David R Nelson; Jared V Goldstone; Joanna Y Wilson Journal: Sci Rep Date: 2021-05-10 Impact factor: 4.379