Characterization, mutagenesis and mechanistic analysis of the sterol C24-methyltransferase: Implications for understanding active site requirements for sterol biosynthesis



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Previous studies that have defined regions of the sterol C24-methyltransferase (SMT) primary structure involved with catalysis failed to show the full identity of essential amino acids associated with the sterol binding segments that contribute to C-methylation from SMTs. Here we report the characterization and inhibition of the C24-SMT from Saccharomyces cerevisiae and Chlamydomonas reinhardtii, a crucial enzyme responsible for the C1 transfer in the 24-alkyl side chain construction of phytosterols. A series of 27 mutants across kingdoms were evaluated for product outcome to locate relevant residues and regions of the SMTs. These mutations converted substrate to product ratios that favored the formation of Δ25(27)-olefins over the preferred Δ24(28)-olefins found in the wild type SMT and revealed a few essential amino acids confirm by a loss of activity. Two transition state-based and product inhibitors evaluated with ScSMT were shown to inhibit SMT activity with IC50 values ranging from 1 µM (25-azalanosterol, a transition state analog) to 7 μM (abasol, a product inhibitor). These IC50 values are similar to antifungals established for opportunistic pathogens. By using inhibitors, sequence alignments of the SMT and site-directed mutagenesis of select residues within the conserved regions of the primary structure, a homology model of Trypanosoma brucei SMT was made that incorporates all of the key structural features of the enzyme in its mechanism of binding to the sterol based on previous binding studies with T. brucei and S. cerevisiae. From the combination of results, the identity of amino acid residues in the active site and inhibition profiles provide a closer look into the catalysis of the sterol C24-SMT.



Sterol, Methyltransferase, Mutagenesis, Inhibitor