EXPLORING MOLECULAR BASIS FOR SUBSTRATE SPECIFICITY OF PHENOLIC O-METHYLTRANSFER

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 木质素是仅次于纤维素的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)上发生甲基化，并被排除在对羟基位置上的取代；事实上，单醇的对羟基被认为对产生氧化自由基和交联木素单元至关重要。因此，单醇的对羟基(即4-OH)位置的甲基化应该会干扰木质素聚合物的合成。为了验证这一假设，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丙烯的区域特异性O-甲基化的分子机制，从而创造一组新的单烯醇基4-O-甲基转移酶，从而在植物中引入非天然的对甲氧基单醇。具体地说，我们将探索两类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。所获得的信息将用于建立木质素3/5-O-甲基转移酶和苯丙烯4-O-甲基转移酶变异体的综合文库，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，我们将选择一系列新的变种，能够有效地甲基化不同单酚的对羟基。这些研究将为木质素生物合成的合理操作提供科学依据，以提高生物燃料的生产效率，从而有助于减少我们对石化燃料的依赖。

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.