Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity

Kulothungan Ganapathy, Christopher W. Jones, Camille M. Stephens, Rit Vatsyayan, Julie A. Marshall, W. David Nes

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Two families of sterol C24-methyltransferase (SMT) are responsible for the formation of the ergostane (C1-transfer activity; SMT1) and stigmastane (C2-transfer activity: SMT2) sterol side chains, respectively. The fungal Saccharomyces cerevisiae SMT1 (Erg6p) operates the first C1-transfer in concerted fashion to form a single product whereas the protozoan and plant SMTs are bifunctional capable of catalyzing two sequential, mechanistically distinct C-methylation activities in the conversion of a Δ24-sterol acceptor to diverse doubly alkylated products. Previous mutation of the amino acids of Erg6p at D79, Y81 and E82 afforded C1 or C2-transfer activities typical of the protozoan and plant SMT. In this study, scanning mutagenesis experiments involving a leucine replacement of 52 amino acids in Erg6p followed by substitution of key residues with functionally or structurally similar amino acids indicated that 5 new residues at positions Y192, G217, G218, T219 and Y223 can switch the course of C1-transfer activity to include plant-like C2-transfer activity. The data support a model in which several conserved and non-conserved amino acids located in distinct regions of the Erg6p regulate the course of the C-methylation reaction toward product differences.

Original languageEnglish
Pages (from-to)344-351
Number of pages8
JournalBiochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
Volume1781
Issue number6-7
DOIs
StatePublished - Jun 2008

Keywords

  • Active site
  • Erg6p
  • Fecosterol
  • Saccharomyces cerevisiae
  • Site-directed mutagenesis
  • Sterol catalysis
  • Sterol methyltransferase yeast
  • Zymosterol

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