Enzymatic C-Methylation Reactions of Phytosterol Biosynthesis

在某些方面，所有的甾醇都是相同的，但在其他方面，它们却截然不同。胆固醇、麦角甾醇和谷甾醇是被广泛认识的化合物之一，它们分别与动物、真菌/原生动物和植物的细胞膜有关。胆固醇与麦角甾醇和谷甾醇在结构上的主要区别是在甾醇侧链上的碳-24；C24分别是H、β-CH3或α-C2H5基团。因此，似乎在添加到“胆固醇”侧链上的C24-烷基(S)的大小和方向之间存在某种关联，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-β24-甾醇甲基转移酶的结构和功能具有重要意义，它是植物甾醇生物合成和动态平衡中的关键转烷基酶(S)，因为这些实验所获得的见解有助于设计控制或模拟其活动的方法。人们发现自然界中存在一类结构相似的酶，它们根据相似的机理计划(甾醇C24-烷基化的空间-电子塞子模型)催化来自共同活性部位的甾醇受体分子的连续C-甲基化。通过定点突变实验，确定了活性部位拓扑结构中四个底物结合区中的三个影响C1/C2-活性的补体和产物的多样性。现在，在本项目中，将通过合理的设计和活性检测相结合的方法来创造具有新性能的SMTs。此外，还将确定来自不同领域的SMT的特性。将进行热力学分析，确定针对这种反应类型的底物和抑制剂的C24-甲基化反应的熵和焓成分；这些信息将与酶生成的产物分布相关联。利用蛋白质化学和X射线结晶学，利用合成的氟化类似物和基于机理的失活剂，确定克隆的SMT中活性位点残基的位置，并估计不同生物在其生活史上不同时期的无细胞制剂中的活性SMT酶浓度，以提供关于植物甾醇生物合成的门类特异性信息。广泛影响该项目汇集了许多以植物甾醇和SMT结构、生物化学和进化为中心的不同努力。这个项目提供了一个在本科生和研究生之间与博士后助理和PI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确定SMT活性中心的相关拓扑结构，以影响催化作用。与酶效率的能量学有关的酶进化的标志(降低激活障碍以促进现存酶相对于祖先形式的催化优势)将通过研究系统发育上不同的SMT首次得到解决。该项目将通过教育学生产生其他更广泛的影响，为他们提供生物化学、分子生物学和天然产品研究中常用的指导和实验方法。

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.