Paper Citing NAMD - Abstract
Morrison, Ann Michelle Stanley; Goldstone, Jared V.; Lamb, David C.; Kubota, Akira; Lemaire, Benjamin; Stegeman, John J.
Identification, modeling and ligand affinity of early deuterostome CYP51s, and functional characterization of recombinant zebrafish sterol 14 alpha-demethylase
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1840:1825-1836, JUN 2014
Background: Sterol 14 alpha-demethylase (cytochrome P450 51, CYP51, P450(14DM)) 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 CYP5ls 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 Abudefdufsaxatilis, and the sea urchin Strongylocentrotus putpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14 alpha-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 14 alpha-demethylase activity, at 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave K-s (dissociation constant) values of 0.26 mu M for ketoconazole and 0.64 mu M 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 CYP5ls 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. (C) 2014 Elsevier B.V. All rights reserved.
