Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

Maristela Pereira, Zhihong Song, Ludier Kesser Santos-Silva, Mathew H. Richards, Thi Thuy Minh Nguyen, Jia Lin Liu, Celia Maria de Almeida Soares, Aline Helena da Silva Cruz, Kulothungan Ganapathy, W. David Nes

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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 42,502Da. 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.

Original languageEnglish
Pages (from-to)1163-1174
Number of pages12
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1801
Issue number10
DOIs
StatePublished - Oct 2010

Keywords

  • Antifungal drug
  • Cloning
  • Erg6p
  • Ergosterol
  • Ergosterol biosynthesis inhibitor
  • Ergosterol evolution
  • Lanosterol catalysis
  • Membrane
  • Paracoccidiodes brasiliensis brassicasterol
  • Sterol metabolome
  • Sterol methylation mechanism

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    Pereira, M., Song, Z., Santos-Silva, L. K., Richards, M. H., Nguyen, T. T. M., Liu, J. L., de Almeida Soares, C. M., da Silva Cruz, A. H., Ganapathy, K., & Nes, W. D. (2010). Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1801(10), 1163-1174. https://doi.org/10.1016/j.bbalip.2010.06.007