Enzymatic C-Methylation Reactions of Phytosterol Biosynthesis

摘要(S)-腺苷-L-蛋氨酸-D24-雌三醇甲基转移酶(SMT)通过植物特有的类固醇途径环青藤醇到谷甾醇来控制碳流。尽管SMT在物理性质、反应复杂性和底物选择性上可能不同，但一类三维相似的SMT被认为存在于植物中，根据空间-电塞模型中设想的类似的机理计划，从共同的活性中心催化甾醇受体分子的连续C-甲基化。活性中心地形上的微小差异被认为是为了确定C1/C2-活性和产物多样性的互补性。此外，通过管理SMT催化对作物中的甾醇途径进行生物工程被认为是带来增值的途径。基于最近NSF支持的从大豆克隆的SMT1和从拟南芥克隆的SMT2的纯化和性质研究的成功，本提案将研究活性中心的相关拓扑结构以及来自不太先进和更先进的植物系统的一系列天然和突变的SMT的性质。本研究的具体目标是：(I)通过化学和光亲和标记技术确定大豆和拟南芥SMT1和SMT2活性中心中的甾醇和ADOMet结合位点和催化残基；(Ii)通过定点诱变实验确定与底物可接受性和产物多样性相关的氨基酸；(Iii)克隆、测序、在大肠杆菌中表达并鉴定产生具有D25(27)-甾醇侧链的植物甾醇侧链的原生生物SMT1的性质；以及(Iv)在合作者的帮助下确定和比较SMT1和SMT2型酶的三维结构。更广泛的影响从SMT酶特性和重新设计实验中获得的知识将为理解C-甲基化催化的机制提供一个框架，并将产生利用定制的新型SMT酶来产生抗虫性的酶功能增益(过表达)植物。这项工作计划将被整合到现有的化学课程中，以获得研究学分，并将涉及到邻近的一所非博士学位授予大学的学生。

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.