516-28-9Relevant academic research and scientific papers
Sterol C24-methyltransferase: Physio- and stereo-chemical features of the sterol C3 group required for catalytic competence
Howard, Alicia L.,Liu, Jialin,Elmegeed, Gamal A.,Collins, Emily K.,Ganatra, Kalgi S.,Nwogwugwu, Chizaram A.,Nes, W. David
, p. 43 - 50 (2012/08/07)
Sterol C24-methyltransferases (24-SMTs) catalyze the electrophilic alkylation of Δ24-sterols to a variety of sterol side chain constructions, and the C3- moiety is the primary determinant for substrate binding by these enzymes. To determine what specific structural features of the C3-polar group ensure sterol catalysis, a series of structurally related C3-analogs of lanosterol that differed in stereochemistry, bulk and electronic properties were examined against the fungal 24-SMT from Paracoccidioides brasiliensis (Pb) which recognize lanosterol as the natural substrate. Analysis of the magnitude of sterol C24-methylation activity (based on the kinetic constants of Vmax/Km and product distributions determined by GC-MS) resulting from changes at the C3-position in which the 3β-OH was replaced by 3α-OH, 3β-acetyl, 3-oxo, 3-OMe, 3β-F, 3β-NH2 (protonated species) or 3H group revealed that lanosterol and five substrate analogs were catalyzed and yielded identical side chain products whereas neither the 3H- or 3α-OH lanosterol derivatives were productively bound. Taken together, our results demonstrate a chemical complementarity involving hydrogen bonding formation of specific active site contacts to the nucleophilic C3-group of sterol is required for proper orientation of the substrate C-methyl intermediate in the activated complex.
Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis
Pereira, Maristela,Song, Zhihong,Santos-Silva, Ludier Kesser,Richards, Mathew H.,Nguyen, Thi Thuy Minh,Liu, JiaLin,de Almeida Soares, Celia Maria,da Silva Cruz, Aline Helena,Ganapathy, Kulothungan,Nes, W. David
body text, p. 1163 - 1174 (2011/11/05)
The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a Kmapp of 38μM and kcatapp of 0.14min-1. Quite unexpectedly, "plant" cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-13C]- or [24-2H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of 1H and 13CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the δ24(25)-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (Ki 14nM) and 26,27-dehydrolanosterol (Ki 54μM and kinact of 0.24min-1) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC50, 30nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C1-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.
EFFECT ON ERGOSTEROL BIOSYNTHESIS OF A FUNGICIDE, SSF-109, IN BOTRYTIS CINEREA
Shirane, Noboru,Murabayashi, Akira,Masuko, Michio,Uomori, Atsuko,Yoshimura, Yohko,et al.
, p. 2513 - 2520 (2007/10/02)
Treatment of Botrytis cinerea with a novel fungicide SSF-109, (dl)-cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)cyclo-heptanol (0.45 μg ml-1), gave five 14α-methyl sterols, 24-methylene-24(25)-dihydrolanosterol, 24-methylene-24(25)-dihydrolanosten-3-one, obtusifoliol, obtusifolione, and 14α-methylfecosterol, together with ergosterol and ergosta-5,8,22-trien-3β-ol.SSF-109 was found to inhibit the biosynthesis of ergosterol at the 14α-demethylation step.
STEROL METHYLTRANSFERASE FROM UROMYCES PHASEOLI: AN INVESTIGATION OF THE FIRST AND THE SECOND TRANSMETHYLATION REACTIONS
Bansal, Surendra K.,Knoche, Herman W.
, p. 1269 - 1278 (2007/10/02)
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.
