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TbSMT [Trypanosoma brucei 24-SMT (sterol C-24- methyltransferase)] synthesizes an unconventional 24-alkyl sterol product set consisting of Δ24(25)-, Δ24(28)- and Δ 25(27)-olefins. The C-methylation reaction requires Si(β)-face C-24-methyl addition coupled to reversible migration of positive charge from C-24 to C-25. The hydride shifts responsible for charge migration in formation of multiple ergostane olefin isomers catalysed by TbSMT were examined by incubation of a series of sterol acceptors paired with AdoMet (S-adenosyl- L-methionine). Results obtained with zymosterol compared with the corresponding 24-2H and 27-13C derivatives revealed isotopic-sensitive branching in the hydride transfer reaction on the path to form a 24-methyl-Δ24(25)-olefin product (kinetic isotope effect, kH/kD =1.20), and stereospecific CH3→ CH2 elimination at the C28 branch and C27 cis-terminal methyl to form Δ24(28)and Δ25(27) products respectively. Cholesta-5,7,22,24-tetraenol converted into ergosta-5,7,22,24(28)-tetraenol and 24β-hydroxy ergosta-5,7,23-trienol (new compound), whereas ergosta-5,24-dienol converted into 24-dimethyl ergosta-5,25(27)-dienol and cholesta-5,7,24-trienol converted into ergosta-5,7,25(27)-trienol, ergosta-5,7,24(28)-trienol, ergosta-5,7,24-trienol and 24 dimethyl ergosta-5,7,25(27)-trienol. We made use of our prior research and molecular modelling of 24-SMT to identify contact amino acids that might affect catalysis. Conserved tyrosine residues at positions 66, 177 and 208 in TbSMT were replaced with phenylalanine residues. The substitutions generated variable loss of activity during the course of the first C-1-transfer reaction, which differs from the corresponding Erg6p mutants that afforded a gain in C-2-transfer activity. The results show that differences exist among 24-SMTs in control of C-1- and C-2-transfer activities by interactions of intermediate and aromatic residues in the activated complex and provide an opportunity for rational drug design of a parasite enzyme not synthesized by the human host. The Authors Journal compilation

Δ(24(28))-Sterols are end products of a mono C-methylation pathway catalyzed by the native Δ(24(25))- to Δ(24(28))-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae. Using a Tyr81 to Phe mutant SMT enzyme of S. cerevisiae, generated by site-directed mutagenesis of a highly conserved residue in the sterol binding site, we found that several Δ(24(25))- and Δ(24(28))-sterols, which are not substrates for the native protein, were catalyzed to mono- and bis-C24-alkylated side chains. The mutant protein behaved similarly to the native protein in chromatography and in binding zymosterol, the preferred substrate. Zymosterol was converted to fecosterol by the Y81F mutant protein with similar turnover efficiency as the native protein (K(m) = 12 μM and k(cat) = 0.01 s-1); trace 24-ethyl sterols were detected from these incubations. 4α-Methyl zymosterol, which is not a normal substrate for the wild-type SMT enzyme, was converted to 4α- methy fecosterol in high yield. When fecosterol and 4α-methyl fecosterol were assayed individually at saturating concentrations only fecosterol served as an effective substrate for the second C-transfer step (K(m) = 38 μM and k(cat) = 0.002 s-1), suggesting that successive C-methylation of Δ(24(28))-substrates is limited by product release and that molecular recognition of sterol features involves hydrogen bond formation. Isomeric 24- ethyl sterol olefins generated from 24(28)-methylene cholesterol were characterized by chromatographic (GC and HPLC) and spectral methods (MS and 1H NMR), viz., fucosterol, isofucosterol, and clerosterol. Changes in rate of C-methylation and product distributions resulting from deuterium substitution at C28 were used to establish the kinetic isotope effects (KIEs) for the various deprotonations leading to C24-methylene, C24-ethylidene, and C24-ethyl sterols. An isotope effect on C28 methyl deprotonation generated during the first C1-transfer was detected with zymosterol and desmosterol paired with AdoMet and [2H3-methyl]AdoMet. A similar experiment to test for a KIE generated during the second C1-transfer reaction with AdoMet paired with 24(28)-methylenecholesterol and [28-2H2]24(28)-methylene cholesterol indicated an inverse isotope effect associated with C27 deprotonation. Alteration in the proportion of the C24 alkylated olefinic products generated by the pure Y81F mutant resulted from the suppression of the formation of Δ(24(28))-ethylidene sterols (C28 deprotonation) by a primary deuterium isotope effect with a compensating stimulation of the formation of 24-ethyl sterols (C27 deprotonation). Kinetic study on the rate of product formation indicated a normal KIE of k(H)/k(D) = 2.62 for the first C1-transfer. Alternatively, an inverse KIE was established with k(H)/k(D) = 0.9 for the second C1-transfer resulting from conversion of the 24(28)-double bond (sp2 hybridization) to a 24β-ethyl group (sp3 hybridization). From the structures and stereochemical assignments of the C-ethyl olefin products, the stereochemistry of the attack of AdoMet in the second C1-transfer was found to operate a Si-face (backside) attack at C24, analogous to the first C1- transfer reaction.

The first syntheses of sterol biosynthetic intermediates zymosterol(4), 4,4-dimethylzymosterol(5), cholesta-8,14,24-trien-3β-ol(6), the 4,4-dimethyl analogue 7, and fecosterol (8) are described in detail. Multigram quantities of key intermediates 16 and 17 were efficiently prepared from known enones 20 and 21 (eight steps, 35% overall yield). Novel entry into Δ8-sterols was achieved through regiospecific hydroboration/deoxygenation of the 8,14-diene systems. Sterols containing Δ24- or Δ(24(28))-olefins were obtained from C24-hydroxy intermediates either via dehydration using bis[α,α-bis(trifluoromethyl)benzenemethanolato]diphenylsulfur in CH2Cl2 or via Swern oxidation/Wittig olefination, respectively. In this way, 16 and 17 were converted to the desired Δ8,24-, Δ8,14,24-, and Δ(8,24(28))-sterols with high regiocontrol.

Key Word Index-Uromyces phaseoli; Pucciniacetate; Basidiomycete; phytosterol biosynthesis; sterol methyltransferase.Abstract-The two-carbon unit at C-24 of many plant, algal and fungal sterols is known to be synthesized by two successive transmethylations with S-adenosylmethionine as the carbon donor.Enzyme(s) for the two transmethylations were isolated from Uromyces phaseoli in an attempt to determine if the two-step reaction is catalysed by separable enzymic activities.The sensitivity of the methyltransferase assay was improved and this modification removed the apparent inhibition of the enzyme(s) by detergents.Several substrates for the second transmethylation step were synthesized by a route developed to convert Δ24-sterols to 24-methylene sterols.The enzymes for the two transmethylation reactions were solubilized and purified.Both activities were purified together approximately the same degree, 155-fold.Degradation products from zymosterol were found to inhibit both reaction steps equally.The data suggest that a single enzyme or complex is responsible for the catalysis of both steps.