Molecular probing of the Saccharomyces cerevisiae sterol 24-C methyltransferase reveals multiple amino acid residues involved with C2-transfer activity.
Two families of sterol C24-methyltransferase (SMT) are responsible for the formation of the ergostane (C(1)-transfer activity; SMT1) and stigmastane (C(2)-transfer activity: SMT2) sterol side chains, respectively. The fungal Saccharomyces cerevisiae SMT1 (Erg6p) operates the first C(1)-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 Delta(24)-sterol acceptor to diverse doubly alkylated products. Previous mutation of the amino acids of Erg6p at D79, Y81 and E82 afforded C(1) or C(2)-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 C(1)-transfer activity to include plant-like C(2)-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.
Ganapathy
Kulothungan
K
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409-1061, USA.
Jones
Christopher W
CW
Stephens
Camille M
CM
Vatsyayan
Rit
R
Marshall
Julie A
JA
Nes
W David
WD
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D015335:Molecular Probes; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase