Mechanism-based Enzyme Inactivators of Phytosterol Biosynthesis

Abstract

Current progress on the mechanism and substrate recognition by sterol methyl transferase (SMT), the role of mechanism-based inactivators, other inhibitors of SMT action to probe catalysis and phytosterol synthesis is reported. SMT is a membrane-bound enzyme which catalyzes the coupled C-methylation-deprotonation reaction of sterol acceptor molecules generating the 24-alkyl sterol side chains of fungal ergosterol and plant sitosterol. This C-methylation step can be rate-limiting in the post-lanosterol (fungal) or post-cycloartenol (plant) pathways. A series of sterol analogs designed to impair SMT activity irreversibly have provided deep insight into the C-methylation reaction and topography of the SMT active site and as reviewed provide leads for the development of antifungal agents.

Mechanistic Analysis of a Multiple Product Sterol Methyltransferase Implicated in Ergosterol Biosynthesis in Trypanosoma brucei

“…In similar fashion, 26,27-dehydrolanosterol fails to serve as a suicide substrate against the yeast SMT (5, 9, 20). When TB SMT was treated with a range of concentrations of 26,27-dehydrozymosterol (25-100 M), a rapid, concentration-and time-dependent loss of SMT activity was observed, consistent with our earlier findings of the inhibitor tested with fungal or plant SMTs (9,20). A plot of the inactivation data as log (residual activity) against incubation time for three inhibitor concentrations gave a series of straight lines (supplemental Fig.…”

“…Substrates and Reagents-The preparation, proof of structure, and purification of 26,27-dehydrozymosterol, 25-azalanosterol, 24(R,S),25-epiminolanosterol, and ergosterol have been described (9,(13)(14)(15). Sterol substrates for activity assay were from our steroid collection (13)(14)(15) Trypanosoma brucei SMT Cloning, Expression, and Purification-Sequence data for T. brucei SMT were obtained from the web site of The Institute of Genomic Research (www.tigr.org) using Blast search against SMT from S. cerevisiae.…”

“…SMT is also of interest enzymologically because the reaction that is catalyzed can be bifunctional with the enzyme accepting different substrate olefins (6). In those protozoans that synthesize ergosterol (7), the C-methylation step makes it possible to develop microbial-specific sterol biosynthesis inhibitors to block a critical slow enzyme in the pathway to treat infected individuals (7)(8)(9).…”

Sterol Biosynthesis Inhibitors: Potential for Transition State Analogs and Mechanism‐Based Inactivators Targeted at Sterol Methyltransferase

“…The SMT from each group was shown to catalyze the inhibitor to a similar methylated product. In addition, the SMT1 from soybean, yeast and Trypanosoma was strongly inactivated, with k inact values of 0.3, 1.5 and 0.3 min -1 observed, respectively [20,38,51], whereas the SMT2 from Arabidopsis was inactivated (k inact of 0.60 min -1 ), and the yeast-like Prototheca SMT1 was not inactivated by the inhibitor [51,52]. The variation in the recognition of the inhibitor by the yeast and yeast-like SMTs suggests that subtle differences exist in the topographies of the active sites of these enzymes.…”

Anti-trypanosomatid activity of platinum–sterol hydrazone imidazoline complexes

“…Rational chemotherapeutic approaches are focused on the metabolic differences between these pathogenic microorganisms and mammals. In this regard, membrane sterol biosynthesis is one of the few areas of difference in primary metabolism between mammals and other eukaryotes such as plants, fungi and protozoa [17,18]. While mammals synthesize C27 cholestane-based members of the sterols family, pathogenic fungi, protozoa and plants require the presence of endogenous sterols C28-C29 (typically ergosterol and 24-alkyl analogs) which are essential for survival and growth factors for these organisms [19,20].…”