EXPLORING MOLECULAR BASIS FOR SUBSTRATE SPECIFICITY OF PHENOLIC O-METHYLTRANSFER

探索酚类邻甲基转移底物特异性的分子基础

• 批准号: 8170616
• 负责人: CHANG-JUN LIU
• 金额: $ 0.63万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170616
• 关键词: Anabolism; Biopolymers; Cellulose; Computer Retrieval of Information on Scientific Projects Database; Dependence; Discrimination; Enzymes; Evolution; Funding; Grant; Hydroxyl Radical; Institution; Libraries; Lignin; Methylation; Methyltransferase; Molecular; Polymers; Positioning Attribute; Production; Protein Engineering; Proteins; Research; Research Personnel; Resources; Screening procedure; Source; Structure; Structure-Activity Relationship; Substrate Specificity; Testing; United States National Institutes of Health; Variant; base; crosslink; design; improved; novel

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lignin is the most abundant terrestrial biopolymer after cellulose. Lignin precursors are exclusively methylated at their meta-positions (i.e., 3/5-OH) of the phenyl rings, and are precluded from the substitution at the para-hydroxyl position; in fact, the para-hydroxyls of monolignols are proposed to be critically important for generating oxidative radicals, and cross-linking lignin units. Therefore, methylation of the para-hydroxyl (i.e., 4-OH) position of monolignol should interfere with the synthesis of the lignin polymer. To test this hypothesis, we propose a structure-based protein engineering approach, to investigate the molecular mechanisms of regiospecific O-methylation of lignin precursors and natural phenylpropenes, thereby, to create a set of novel monolignol 4-O-methyltransferases that will introduce the non-natural para-methoxyl monolignols in planta. Specifically, we will explore the structure-function relationships of two types of functionally distinct but structurally related enzymes, viz., phenylpropene 4-O-methyltransferase and lignin 3/5-O-methyltransferase, to understand the distinct regiospecific methylation and substrate discrimination. The resulting information will be used to create comprehensive libraries of the variants of lignin 3/5-O-methyltransferase and phenylpropene 4-O-methyltransferase, employing both the approaches of structure-based rational design and the directed protein evolution. With high-throughput colorimetric screening, we will select a range of novel variants able to efficiently methylate the para-hydroxyl of the different monolignols. Information from these studies will provide a scientific underpinning for the rational manipulation of lignin biosynthesis to improve the efficiency of biofuel production, and thus contribute to decreasing our dependence on petrochemical fuels.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 木质素是继纤维素之后最丰富的陆地生物聚合物。木质素前体仅在苯环的间位（即 3/5-OH）处甲基化，并且排除对位羟基位置处的取代；事实上，木质素单体的对位羟基对于产生氧化自由基和交联木质素单元至关重要。 因此，木质素单体的对位羟基（即4-OH）位置的甲基化会干扰木质素聚合物的合成。 为了检验这一假设，我们提出了一种基于结构的蛋白质工程方法，研究木质素前体和天然苯基丙烯的区域特异性O-甲基化的分子机制，从而创建一组新型木质素单体4-O-甲基转移酶，将非天然对甲氧基木质素单体引入植物中。具体来说，我们将探索两种功能不同但结构相关的酶（即苯丙烯4-O-甲基转移酶和木质素3/5-O-甲基转移酶）的结构-功能关系，以了解不同的区域特异性甲基化和底物区分。由此产生的信息将用于创建木质素 3/5-O-甲基转移酶和苯丙烯 4-O-甲基转移酶变体的综合文库，采用基于结构的合理设计和定向蛋白质进化的方法。 通过高通量比色筛选，我们将选择一系列能够有效甲基化不同木质素单体的对羟基的新型变体。这些研究的信息将为合理操纵木质素生物合成提供科学基础，以提高生物燃料生产效率，从而有助于减少我们对石化燃料的依赖。