Purification, characterization and inhibition of sterol C24-methyltransferase from Candida albicans

Kulothungan Ganapathy, Ragu Kanagasabai, Thi Thuy Minh Nguyen, W. David Nes

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Abstract

Solubilized sterol C24-methyltransferase (24-SMT) was purified to homogeneity from a cell extract of the yeast Candida albicans (Ca) by anion exchange chromatography, gel permeation chromatography and fast performance liquid chromatography using a Mono Q column. The purified enzyme has an apparent molecular mass of 178 kDa on gel permeation chromatography and 43 kDa on SDS/PAGE, indicating that it is composed of four identical subunits. The substrate requirement of the native enzyme has an optimal specificity for zymosterol with associated kinetic constants of Km 50 μM and kcat of 0.01 s-1. The product of the enzyme incubated with zymosterol was fecosterol. Inhibition of the catalyst was observed with substrate analogs designed as transition state analogs (25-azalanosterol, K i = 54 nM and 24 (R,S),25-epiminolanosterol, Ki = 11 nM) or as mechanism-based inactivators (26,27-dehydrozymosterol, Ki 9 μM) and kinact = 0.03 min-1) of the C24-methylation reaction. Product analogs ergosterol and fecosterol, but neither cholesterol nor sitosterol, inhibited activity affording Ki values of 20 and 72 μM, respectively. Ammonium and thia analogs of the intermediates of the sterol C24-methyl reaction sequence were effective growth inhibitors exhibiting IC50 values that ranged from 3 to 20 μM.

Original languageEnglish
Pages (from-to)194-201
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume505
Issue number2
DOIs
StatePublished - Jan 15 2011

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Keywords

  • Antifungal agents
  • Ergosterol biosynthesis
  • Membranes
  • Sterol C24-methyltransferase
  • Sterol inhibitors
  • Suicide substrates

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