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

The ERG6 gene that encodes (S)-adenosyl-L-methionine:Delta(24(25))- to Delta(24(28))-sterol methyl transferase (SMT) enzyme from Saccharomyc es cerevisiae was introduced into plasmid pET23a(+) and the resulting native protein was overexpressed in BL21(DE3) host cells under control of a T-7 promoter. This enzyme was purified to apparent homogeneity b y ammonium sulfate precipitation, anion exchange, and hydrophobic inte raction chromatography. N-Terminal sequence analysis of the first 10 a mino acids of the purified SMT protein confirmed the identity of the s tart triplet and expected primary structure. The enzyme exhibited a tu rnover number of 0.01/s and an isoelectric point of 5.95. A combinatio n of Superose 6 chromatography and sodium dodecyl sulfate-polyacrylami de gel electrophoresis showed that the purified SMT enzyme possessed a native molecular weight of 172,000 and was tetrameric. The purified S MT enzyme generated kinetics in which velocity versus substrate curves relative to zymosterol (preferred sterol acceptor molecule) and AdoMe t were sigmoidal rather: than hyperbolic, indicating enzyme cooperativ ity among the subunits. Studies on product formation using [27-C-13]zy mosterol and [H-2(3)-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 polyp eptide from S. cerevisiae with related sterol methyl transferase enzym es of plant and fungal origin indicate that there is a significant deg ree of similarity between these enzymes. Specifically, there is a cons erved sequence tin 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 methyltransferase enzymes. (C) 1998 Academic Press.