Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei

Sterol methyltransferase (SMT) plays a key role in sterol biosynthesis in different pathogenic organisms by setting the pattern of the side chain structure of the final product. This catalyst, absent in humans, provides critical pathway-specific enzymatic steps in the production of ergosterol in fungi or phytosterols in plants. The new SMT gene was isolated from Trypanosoma brucei genomic DNA and cloned into an Escherichia coli expression system. The recombinant SMT was purified to homogeneity to give a band at 40.0 kDa upon SDS-PAGE and showed a tetrameric subunit organization by gel chromatography. It has a pH optimum of 7.5, an apparent k_cat value of 0.01 s ^-1, and a K_m of 47 ± 4 μM for zymosterol. The products of the reaction were a mixture of C24-monoalkylated sterols, ergosta-8,24 (25)-dienol, ergosta-8,25 (27)-dienol, and ergosta-8,24 (28)-dienol (fecosterol), and an unusual double C24-alkylated sterol, 24,24-dimethyl ergosta-8,25 (27)-dienol, typically found in plants. Inhibitory profile studies with 25-azalanosterol (K_i value of 39 nM) or 24(R,S),25- epiminolanosterol (K_i value of 49 nM), ergosterol (K_i value of 27 μM) and 26,27-dehydrozymosterol (K_i and k _inact values of 29 μM and 0.26 min^-1, respectively) and data showing zymosterol as the preferred acceptor strongly suggest that the protozoan SMT has an active site topography combining properties of the SMT1 from plants and yeast (37-47% identity). The enzymatic activation of this and other SMTs reveals that the catalytic requirements for the C-methyl reaction are remarkably versatile, whereas the inhibition studies provide a powerful approach to rational design of new anti-sleeping sickness chemotherapeutic drugs.