Enzymatic C-Methylation Reactions of Phytosterol Biosynthesis

在某些方面，所有的固醇都是相同的，但在其他方面，它们又大不相同。胆固醇、麦角甾醇和谷甾醇不仅是被广泛认识的化合物，而且通常分别与动物、真菌/原生动物和植物的膜有关。胆固醇与麦角甾醇和谷甾醇结构的主要区别在于其侧链的碳-24；C24分别是H、β - ch3或α - c2h5基团。由此看来，添加到“胆固醇”侧链上的c24 -烷基基团的大小和方向之间存在某种联系，微生物和植物的独特生物学需要不同类型和数量的植物甾醇（24-烷基甾醇）来生长和繁殖。了解(S)-腺苷- l-蛋氨酸- δ ta24-甾醇甲基转移酶（SMT）的结构和功能具有重要意义，因为从这些实验中获得的见解可能有助于设计控制或模拟其作用的方法。SMT是参与植物甾醇生物合成和稳态的关键转烷基化酶。在NSF的支持下，研究人员克隆并鉴定了smt的变体，并研究了一系列带有修饰侧链的甾醇衍生物作为该酶的机制探针。在自然界中发现了一个结构相似的酶家族，它们根据类似的机制计划（甾醇c24 -烷基化的立体电塞模型）催化一个甾醇受体分子从一个共同的活性位点连续进行c-甲基化。通过定点诱变实验，鉴定出活性位点地形中4个底物结合区中的3个影响C1/ c2活性补体和产物多样性。现在，在这个项目中，将通过合理设计和活性分析相结合来创建具有新特性的smt。此外，还将确定来自不同门的smt的特性。热力学分析，确定的熵和焓组分的c24甲基化反应的底物和抑制剂这种反应类型将进行；这些信息将与酶产生的产物分布相关联。利用蛋白质化学和x射线晶体学，利用合成氟化类似物和基于机制的失活剂，确定克隆SMT中活性位点残基的位置，并估计不同生物体在其生活史的不同时间的无细胞制剂中活性SMT酶的浓度，以提供植物甾醇生物合成的门特异性信息。更广泛的影响该项目汇集了许多不同的努力集中在植物甾醇和SMT结构，生物化学和进化。本项目提供了本科生和研究生与博士后助理和项目负责人进行科学概念交流的机会。从SMT表征和重新设计实验中获得的知识将定义SMT活性中心的相关地形，以影响催化。与酶效率的能量学相关的酶进化标志（降低激活障碍以促进现有酶比祖先形式具有催化优势）将首次通过研究系统发育上不同的smt来解决。该项目将通过为学生提供指导和在生物化学、分子生物学和天然产物研究中经常使用的实验方法，对学生进行教育，从而产生其他更广泛的影响。

In certain ways all sterols are the same, yet in other respects, they are quite different. Cholesterol, ergosterol and sitosterol are among the compounds which are not only widely recognized but are also commonly associated with the membranes of animals, fungi/protozoa and plants, respectively. The major difference in the structure of cholesterol with that of ergosterol and sitosterol is at carbon-24 in the sterol side chain; C24 is an H, beta-CH3 or alpha-C2H5 group, respectively. It would appear, therefore, that there is some sort of association between the size and direction of the C24-alkyl group(s) added to the "cholesterol" side chain and that the unique biology of microbes and plants require distinct types and amounts of phytosterols (24-alkyl sterols) to grow and reproduce. Understanding the structure and function of the (S)-adenosyl-L-methionine-delta24-sterol methyltransferase (SMT), the crucial transalkylating enzyme(s) involved in phytosterol biosynthesis and homeostasis, are of significant importance as insights gained from these experiments may facilitate the design of methods to control or mimic their actions.With previous NSF support, variant SMTs were cloned and characterized and a series of sterol derivatives with modified side chains were investigated as mechanistic probes for this enzyme. A family of structurally similar enzymes was discovered to exist in nature, which 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 of sterol C24-alkylation). Through site-directed mutagenesis experiments, three of four substrate binding regions in the active site topography were identified to affect the complement of C1/C2-activities and product diversity. Now, in this project, SMTs with novel properties will be created by a combination of rational design and activity assay. In addition, the properties of SMTs from different phyla across kingdoms will be determined. Thermodynamic analysis that determines the entropic as well as enthalpic components of the C24-methylation reaction toward substrates and inhibitors of this reaction type will be conducted; this information will be correlated to the enzyme-generated product distributions. The location of active site residues in cloned SMTs and an estimation of the active SMT enzyme concentration in cell-free preparations of different organisms at different times in their life history will be determined utilizing protein chemistry and X-ray crystallography utilizing synthetic fluorinated analogs and mechanism-based inactivators to afford phyla-specific information on phytosterol biosynthesis.Broader ImpactsThis project brings together a number of different efforts centered on phytosterol and SMT structure, biochemistry and evolution. This project presents an opportunity for cross-talk of scientific concepts between undergraduate and graduate students with the postdoctoral associates and PI. Knowledge gained from the SMT characterization and redesign experiments will define relevant topographies in the active center of SMTs to affect catalysis. The hallmark of enzyme evolution that relates to the energetics of enzyme efficiency (the lowering of activation barriers to promote catalytic superiority of the extant enzyme over the ancestral form) will be addressed for the first time by studying phylogenetically different SMTs. The project will give rise to other broader impacts through the education of students by providing them mentoring and experimental approaches used routinely in biochemistry, molecular biology and the study of natural products.