Unraveling the Catalytic Specificity of Terpene Hydroxylases and Engineering Sesquiterpene Hydroxylation into Plants

揭示萜烯羟化酶的催化特异性以及将倍半萜烯羟化转化为植物

• 批准号: 0721203
• 负责人: Joseph Chappell
• 金额: $ 56.04万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0721203&HistoricalAwards=false
• 关键词: Unraveling; Catalytic; Specificity; Terpene; Hydroxylases

Terpenes represent a complex array of chemical compounds that are essential for many facets of plant growth and development, are of considerable importance for their nutritional contributions to animals and man, and are an important source of natural products used in agriculture and medicine. Hence, it is not surprising that the biochemistry and molecular biology of terpene bioynthesis has been intensively studied. Nonetheless, mechanistic appreciation for many of the enzymes making up these biosynthetic pathways is still very limited. The current project addresses this drawback by focusing on one particular class of enzymes, terpene hydroxylases, and even more narrowly to the family of sesquiterpene hydroxylases. These enzymes decorate terpene hydrocarbon skeletons with one to several hydroxyl substituents in very specific patterns that impart biological activities to the sesquiterpene compounds and serve as handles for further in vivo modifications. Recent studies suggest that the specificity for these biosynthetic reactions reside within specific regions of the enzymes themselves. Hence, the first objective of the current research is to more precisely define the structural elements of sesquiterpene hydroxylases that regulate and control their catalytic activities. This is to be accomplished by converting the catalytic specificity of one sesquiterpene hydroxylase into that of a closely related hydroxylase based on an iterative and rational mutagenesis program designed upon a combination of structural and molecular comparisons between the two enzymes. The ultimate aim is to create new plant traits, like insect and disease resistance, and the biosynthesis of high-value natural products in plants based upon the manipulation of terpene metabolism in plants. Therefore, additional work will further recent advances in the genetic engineeringof sesquiterpene metabolism by comparing strategies for introducing expression of unique terpene hydroxylases in transgenic plants, and evaluating these plants for the biosynthesis of new, biologically active sesquiterpenes.Broader impacts: The current project represents a strong interdisciplinary effort between chemists, biologists, biochemists, and molecular biologists, involving undergraduate and graduate students, postdoctoral associates, and especially persons from US populations under-represented in the sciences, in an effort to provide new insights into how metabolic pathways might be engineered for enhanced value using emerging technologies.

萜烯是一系列复杂的化合物，对植物生长和发育的许多方面都至关重要，对动物和人类的营养贡献相当重要，是农业和医药中使用的天然产物的重要来源。因此，萜类生物合成的生物化学和分子生物学得到了广泛的研究也就不足为奇了。尽管如此，对构成这些生物合成途径的许多酶的机理认识仍然非常有限。目前的项目通过关注一类特殊的酶，萜烯羟化酶，甚至更狭隘地关注倍半萜烯羟化酶家族来解决这个缺点。这些酶以一到几个羟基取代基以非常特定的模式修饰萜烯碳氢化合物骨架，赋予倍半萜化合物生物活性，并作为进一步体内修饰的手柄。最近的研究表明，这些生物合成反应的特异性存在于酶本身的特定区域。因此，当前研究的第一个目标是更精确地定义调节和控制其催化活性的倍半萜羟化酶的结构元素。这是通过将一种倍半萜羟化酶的催化特异性转化为另一种密切相关的羟化酶的催化特异性来实现的，这是基于两种酶的结构和分子比较组合设计的迭代和合理的诱变程序。最终目标是创造新的植物性状，如抗虫和抗病，以及基于植物萜类代谢的植物高价值天然产物的生物合成。因此，通过比较在转基因植物中引入独特萜烯羟化酶的表达策略，以及评价这些植物生物合成新的、具有生物活性的倍半萜的能力，将进一步推动倍半萜代谢基因工程的最新进展。更广泛的影响：目前的项目代表了化学家、生物学家、生物化学家和分子生物学家之间强有力的跨学科努力，涉及本科生和研究生、博士后助理，特别是来自美国科学界代表性不足的人群，旨在为如何利用新兴技术设计代谢途径以提高价值提供新的见解。