Enzymatic C-Methylation Reactions in Phytosterol Biosynthesis

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Sterol C-methyl transfer reactions catalyzed by the (S)-adenosyl-L-methionine: .24 -sterol methyl transferase (SMT) control the production and processing of sitosterol biosynthesis in plants. SMTs differ in physical properties, reaction complexity and the complement of C1/C2-activities. This project tests the proposal that a family of three-dimensionally similar SMT isoforms exists in plants and catalyzes the successive C-methylations of a sterol acceptor molecule from a common active site according to a similar mechanistic plan (the steric-electric plug model). Minor differences in the active site topography are considered to determine the complement of C-methylation activities and product outcome. In this project, cloned SMTs from Arabidopsis thaliana and Glycine max will be produced at high levels. The specific goals of this project are:(1) Characterization of the kinetic mechanism and product identities involved with the first and second C1-transfer reactions. For this purpose, substrate analogs and ATP will be used to probe catalytic action of the recombinant SMTs and GC-MS and NMR will be employed in product structure determination.(2) Identification of the sterol- and AdoMet-binding sites and catalytic amino acids in the active center by chemical and photoaffinity labeling techniques. Information from the active site location, plus that gained by primary sequence comparisons with related SMTs, will be used to guide site-directed mutagenesis of select amino acid residues in the active center to generate unusual product diversity that can benefit the plant defense system. Knowledge gained from this research is expected to be broadly applicable to the understanding of molecular biochemistry and biosynthetic investigations in general.