TY - JOUR
T1 - Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C-2-transfer activity
AU - Ganapathy, Kulothungan
AU - Jones, Christopher W.
AU - Stephens, Camille M.
AU - Vatsyayan, Rit
AU - Marshall, Julie A.
AU - Nes, William
N1 - Funding Information:
This study was supported by the National Science Foundation (MCB 0417436) and Welch Foundation (D-1276).
PY - 2008/6
Y1 - 2008/6
N2 - 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.
AB - 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.
KW - Active site
KW - Erg6p
KW - Fecosterol
KW - Saccharomyces cerevisiae
KW - Site-directed mutagenesis
KW - Sterol catalysis
KW - Sterol methyltransferase yeast
KW - Zymosterol
UR - http://www.scopus.com/inward/record.url?scp=50949132701&partnerID=8YFLogxK
U2 - 10.1016/j.bbalip.2008.04.015
DO - 10.1016/j.bbalip.2008.04.015
M3 - Article
C2 - 18503783
SN - 1388-1981
VL - 1781
SP - 344
EP - 351
JO - Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids
JF - Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids
IS - 6-7
ER -