Cloning,mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis |
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Authors: | Maristela Pereira Zhihong Song Ludier Kesser Santos-Silva Mathew H. Richards Thi Thuy Minh Nguyen JiaLin Liu Celia Maria de Almeida Soares Aline Helena da Silva Cruz Kulothungan Ganapathy W. David Nes |
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Affiliation: | 1. Laboratorio de Biologia Molecular, Instituto de Ciencias Biologicas, Universidade Federal de Goias, 74001-970, Goiania, Goias, Brazil;2. Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA |
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Abstract: | 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 42,502 Da. The enzymatic C24-methylation gave a Kmapp of 38 μM and kcatapp of 0.14 min−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 14 nM) and 26,27-dehydrolanosterol (Ki 54 μM and kinact of 0.24 min−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, 30 nM) 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. |
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Keywords: | SMT, Sterol C24-methyltransferase GC-MS, Gas-chromatography-mass spectroscopy Pb, Paracoccidiodes brasiliensis Sc, Saccharomyces cerevisiae |
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