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 3.2 nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26 μM for ketoconazole and 0.64 μM 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 CYP51. 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.

Genetic and structural analyses of cytochrome P450 hydroxylases in sex hormone biosynthesis: Sequential origin and subsequent coevolution

“…Interestingly, despite the lack of clear steroidogenic CYP orthologs in tunicates, several ovarian steroids have been identified in Ciona intestinalis (Cangialosi et al, 2010). However, as we recently noted, there is no lanosterol 14-a demethylase (CYP51) in either of the Ciona species’ genomes (Morrison et al, 2014). CYP51 is required for de novo cholesterol synthesis leading to an obligate dietary or commensal source for steroid precursors in Ciona spp.…”

“…In fact, the genomes of the ascidian tunicate species Ciona intestinalis and Ciona savignyi lack CYP51 (lanosterol demethylase), the enzyme required for the biosynthesis of the steroid precursor cholesterol (Morrison et al, 2014). CYP51 also appears to be missing from the genomes of the appendicularian Oikopleura dioica and the molgulid Molgula occidentalis , suggesting that CYP51 was lost early in tunicate evolution.…”

Occurrence and origin of sensitivity toward difenoconazole in zebrafish (Danio reio) during different life stages

“…Morrison et al (2014) found that ketoconazole has the potential to inhibit zebrafish CYP51, based on the results of a docking experiment. Skolness et al (2013) demonstrated that propiconazole could inhibit steroidogenesis in the fathead minnow (Pimephales promelas) and down-regulated the expression of some key genes, such as CYP51, HMGCR and FDPS, in the hepatic synthesis process.…”

Design, Synthesis Molecular Docking Study and Antifungal Activity Evaluation of New Benzimidazole-Triazole Derivatives

“…Selective inhibition of the enzyme would cause depletion of ergosterol and accumulation of lanosterol and result in the growth inhibition of the fungal cell [1].In this study, a series of benzimidazole derivatives containing a triazole ring that are structurally related to the famous antifungal azole pharmacophore were synthesized and their structures were characterized by spectral (IR, 1 H-NMR, 13 C-NMR, and MS spectra) analyses. Compounds 5i and 5s showed the most promising antifungal activity with a MIC50 value of 0.39 ug/mL against Candida species.…”

“…Selective inhibition of the enzyme would cause depletion of ergosterol and accumulation of lanosterol and result in the growth inhibition of the fungal cell [1].…”