Enzymatic C-Methylation Reactions in Phytosterol Biosynthesis

由(S)-腺苷-L-蛋氨酸：24-甾醇甲基转移酶(SMT)催化的甾醇C-甲基转移反应控制植物谷甾醇的生物合成。SMT的物理性质、反应复杂性和C1/C2-活性的互补性不同。这个项目测试了植物中存在一系列三维相似的SMT异构体的假设，并根据类似的机制计划(空间-电塞模型)催化来自共同活性部位的甾醇受体分子连续的C-甲基化。活性部位的微小差异被认为是决定C-甲基化活性和产物结果的补充性。在这个项目中，将高水平地生产从拟南芥和大豆中克隆的SMT。该项目的具体目标是：(1)表征与第一和第二个氯转移反应有关的动力学机制和产物特性。为此，将使用底物类似物和ATP来探索重组SMT的催化作用，并将GC-MS和核磁共振用于产物结构的确定。(2)利用化学和光亲和标记技术鉴定活性中心中的甾醇和Adobe Met结合部位和催化氨基酸。来自活性中心位置的信息，加上通过与相关SMT进行初级序列比较获得的信息，将被用于指导活性中心精选氨基酸残基的定点突变，以产生有利于植物防御系统的不寻常的产物多样性。从这项研究中获得的知识有望广泛适用于对分子生物化学和生物合成研究的一般理解。

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.