Poster: Lipids & Related Molecules
Abs #
864: Sterol Methyl Transferase: Variability of Kinetic Mechanism and Similarity of Reaction Mechanism in Soybean, Arabidopsis and Yeast Isoforms
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Presenter: |
Nes, William David, wdavid.nes@ttu.edu |
Authors | Nes, William David (A) Zhou, Wenxu (A) Song, Zhihong (A) Dennis, Allen L (A) Miller, Mathew B (A) | | Affiliations: |
(A): Texas Tech University
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Sterol C-methylations catalyzed by the (S)-adenosyl-L-methionine:C-24-sterol methyltransferase (SMT) have provided the focus for study of electrophilic alkylations, a reaction type of functional importance in C-C bond formation of natural products. SMTs occur generally in nature, but do not occur in animal systems, suggesting that the difference in sterol synthetic pathways can exploited therapeutically and in insect-plant interactions. In this study, we cloned SMTs from I. Glycine max, II. Arabidopsis thaliana and III. Saccharomyces cerevisiae and purified the resulting recombinant enzyme in native form to test the proposal that each of these enzymes can bind and transform Delta-24-sterols according to a similar mechanistic plan. Kinetic and inhibition studies with I, II and III indicated that the Michaelis constants calculated for AdoMet and sterol were similar (ca. 50 ± 20 µM) but the binding order for the substrates and the sterol specificity (measured as catalytic competence: kcat/Km) differed as well as turnover numbers for the different sterol side chains- Delta-24(25)- versus Delta-24(28). From the relative reaction rates of native and mutant enzymes modified in the sterol binding site (within the ERG6 signature motif Y81EYGWG86) and assayed with 27-13C-labeled sterols it was deduced that the alignment of the nucleophilic centers at C-3 and C-24 on the acceptor molecule relative to the binding pocket containing AdoMet was essential for C1/C2-activity. Substrate channeling was found to operate by the same si-face
mechanism to produce single or multiple 24-alkyl olefins. From the combination of results, the interrelationships of substrate functional groups and product outcomes could be approximated and the topology of binding to the SMT thereby formulated.
