Overexpression, purification, and stereochemical studies of the recombinant (S)-adenosyl-L-methionine: delta 24(25)- to delta 24(28)-sterol methyl transferase enzyme from Saccharomyces cerevisiae

Abstract

The ERG6 gene that encodes (S)-adenosyl-L-methionine: delta 24(25)-to delta 24(28)-sterol methyl transferase (SMT) enzyme from Saccharomyces cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21 (DE3) host cells under control of a T7 promoter. This enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, anion exchange, and hydrophobic interaction chromatography. N-Terminal sequence analysis of the first 10 amino acids of the purified SMT protein confirmed the identity of the start triplet and expected primary structure. The enzyme exhibited a turnover number of 0.01/s and an isoelectric point of 5.95. A combination of Superose 6 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the purified SMT enzyme possessed a native molecular weight of 172,000 and was tetrameric. The purified SMT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMet were sigmoidal rather than hyperbolic, indicating enzyme cooperativity among the subunits. Studies on product formation using [27-13C]zymosterol and [2H3-methyl]AdoMet incubated with the pure SMT enzyme confirmed the reaction mechanism of sterol methylation to involve a 1,2-hydride shift of H-24 to C-25 from the Re-face of the original 24,25- double bond. Deduced amino acid sequence comparisons of the SMT polypeptide from S. cerevisiae with related sterol methyl transferase enzymes of plant and fungal origin indicate that there is a significant degree of similarity between these enzymes. Specifically, there is a conserved sequence (in yeast from amino acids ca. 79 to 92 which contains an YEXGWG motif; referred to as Region I) that is not present in other AdoMet-dependent methyl transferase enzymes.