A nematode sterol C4α-methyltransferase catalyzes a new methylation reaction responsible for sterol diversity.

Primitive sterol evolution plays an important role in fossil record interpretation and offers potential therapeutic avenues for human disease resulting from nematode infections. Recognizing that C4-methyl stenol products [8(14)-lophenol] can be synthesized in bacteria while C4-methyl stanol products (dinosterol) can be synthesized in dinoflagellates and preserved as sterane biomarkers in ancient sedimentary rock is key to eukaryotic sterol evolution. In this regard, nematodes have been proposed to convert dietary cholesterol to 8(14)-lophenol by a secondary metabolism pathway that could involve sterol C4 methylation analogous to the C2 methylation of hopanoids (radicle-type mechanism) or C24 methylation of sterols (carbocation-type mechanism). Here, we characterized dichotomous cholesterol metabolic pathways in Caenorhabditis elegans that generate 3-oxo sterol intermediates in separate paths to lophanol (4-methyl stanol) and 8(14)-lophenol (4-methyl stenol). We uncovered alternate C3-sterol oxidation and Δ7 desaturation steps that regulate sterol flux from which branching metabolite networks arise, while lophanol/8(14)-lophenol formation is shown to be dependent on a sterol C4α-methyltransferse (4-SMT) that requires 3-oxo sterol substrates and catalyzes a newly discovered 3-keto-enol tautomerism mechanism linked to S-adenosyl-l-methionine-dependent methylation. Alignment-specific substrate-binding domains similarly conserved in 4-SMT and 24-SMT enzymes, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of methyl sterols. The combination of these results provides evolutionary leads to sterol diversity and points to cryptic C4-methyl steroidogenic pathways of targeted convergence that mediate lineage-specific adaptations.-.

Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications.

Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via Δ24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via Δ25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation-π interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.

Binding of a physiological substrate causes large-scale conformational reorganization in cytochrome P450 51.

Sterol 14α-demethylases (CYP51s) are phylogenetically the most conserved cytochromes P450, and their three-step reaction is crucial for biosynthesis of sterols and serves as a leading target for clinical and agricultural antifungal agents. The structures of several (bacterial, protozoan, fungal, and human) CYP51 orthologs, in both the ligand-free and inhibitor-bound forms, have been determined and have revealed striking similarity at the secondary and tertiary structural levels, despite having low sequence identity. Moreover, in contrast to many of the substrate-promiscuous, drug-metabolizing P450s, CYP51 structures do not display substantial rearrangements in their backbones upon binding of various inhibitory ligands, essentially representing a snapshot of the ligand-free sterol 14α-demethylase. Here, using the obtusifoliol-bound I105F variant of Trypanosoma cruzi CYP51, we report that formation of the catalytically competent complex with the physiological substrate triggers a large-scale conformational switch, dramatically reshaping the enzyme active site (3.5-6.0 Å movements in the FG arm, HI arm, and helix C) in the direction of catalysis. Notably, our X-ray structural analyses revealed that the substrate channel closes, the proton delivery route opens, and the topology and electrostatic potential of the proximal surface reorganize to favor interaction with the electron-donating flavoprotein partner, NADPH-cytochrome P450 reductase. Site-directed mutagenesis of the amino acid residues involved in these events revealed a key role of active-site salt bridges in contributing to the structural dynamics that accompanies CYP51 function. Comparative analysis of apo-CYP51 and its sterol-bound complex provided key conceptual insights into the molecular mechanisms of CYP51 catalysis, functional conservation, lineage-specific substrate complementarity, and druggability differences.

Functional importance for developmental regulation of sterol biosynthesis in Acanthamoeba castellanii.

The sterol metabolome of Acanthamoeba castellanii (Ac) yielded 25 sterols. Substrate screening of cloned AcCYP51 revealed obtusifoliol as the natural substrate which converts to ∆8,14-sterol (<95%). The combination of [2H3-methyl]methionine incubation to intact cultures showing C28-ergosterol incorporates 2-2H atoms and C29-7-dehydroporiferasterol incorporates 5 2H-atoms, the natural distribution of sterols, CYP51 and previously published sterol methyltransferase (SMT) data indicate separate ∆24(28)- and ∆25(27)-olefin pathways to C28- and C29-sterol products from the protosterol cycloartenol. In cell-based culture, we observed a marked change in sterol compositions during the growth and encystment phases monitored microscopically and by trypan blue staining; trophozoites possess C28/C29-∆5,7-sterols, viable encysted cells (mature cyst) possess mostly C29-∆5-sterol and non-viable encysted cells possess C28/C29-∆5,7-sterols that turnover variably from stress to 6-methyl aromatic sterols associated with changed membrane fluidity affording lysis. An incompatible fit of steroidal aromatics in membranes was confirmed using the yeast sterol auxotroph GL7. Only viable cysts, including those treated with inhibitor, can excyst into trophozoites. 25-Azacycloartanol or voriconazole that target SMT and CYP51, respectively, are potent enzyme inhibitors in the nanomolar range against the cloned enzymes and amoeba cells. At minimum amoebicidal concentration of inhibitor amoeboid cells rapidly convert to encysted cells unable to excyst. The correlation between stage-specific sterol compositions and the physiological effects of ergosterol biosynthesis inhibitors suggests that amoeba fitness is controlled mainly by developmentally-regulated changes in the phytosterol B-ring; paired interference in the ∆5,7-sterol biosynthesis (to ∆5,7) - metabolism (to ∆5 or 6-methyl aromatic) congruence during cell proliferation and encystment could be a source of therapeutic intervention for Acanthamoeba infections.