Enzymatic C-Methylation Reactions of Phytosterol Biosynthesis

点击翻译按钮获取中文摘要

ABSTRACT(S)-adenosyl-L-methionine-D24-strerol methyltransferase (SMT) controls carbon flux through the plant-specific sterol pathway cycloartenol to sitosterol. Despite the fact that SMTs can differ in physical properties, reaction complexity and substrate selectivity, a family of three-dimensionally similar SMTs are proposed to exist in plants catalyzing the successive C-methylations of a sterol acceptor molecule from a common active site according to a similar mechanistic plan as envisaged in the steric-electric plug model. Minor differences in the active site topography are considered to determine the complement of C1/C2- activities and product diversity. Additionally, bioengineering the sterol pathway in crops by managing SMT catalysis is considered to lead to value-added traits.Based on recent success from NSF supported research in purifying and characterizing the properties of cloned SMT1 from soybean and SMT2 from Arabidopsis, the present proposal will investigate the relevant topographies of the active center and properties of a series of native and mutant SMTs from less-advanced and more-advanced plant systems. The specific objectives are to (i) identify the sterol and AdoMet-binding sites and catalytic residues in the active center of SMT1 and SMT2 from soybean and Arabidopsis by chemical and photoaffinity labeling techniques; (ii) determine amino acids involved with substrate acceptability and product diversity through site-directed mutagenesis experiments; (iii) clone, sequence, express in E. coli and characterize the properties of the Prototheca wickerhamii SMT1, which generates a novel phytosterol side chain possessing the D25(27) -sterol side chain; and (iv) define and compare the three-dimensional structures of SMT1- and SMT2-type enzymes with the assistance of collaborators. Broader ImpactsKnowledge gained from SMT enzyme characterization and redesign experiments will provide a framework for understanding the mechanism of C-methylation catalysis and will generate gain-in-enzyme-function (overexpression) plants engineered with novel SMT enzymes tailored to generate insect resistance. The work plan will be integrated into existing chemistry courses for research credit and will involve outreach to students at a neighboring non Ph.D. degree granting university.