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

萜烯代表了一系列复杂的化合物，它们对植物生长和发育的许多方面都是必不可少的，对于它们对动物和人类的营养贡献具有相当重要的意义，并且是用于农业和医学的天然产物的重要来源。因此，萜烯生物合成的生物化学和分子生物学被深入研究并不奇怪。尽管如此，对构成这些生物合成途径的许多酶的机械认识仍然非常有限。目前的项目通过关注一类特定的酶，萜烯羟化酶，甚至更狭义地关注倍半萜烯羟化酶家族来解决这个缺点。这些酶以非常特定的模式用一个至几个羟基取代基修饰萜烯烃骨架，所述羟基取代基赋予倍半萜化合物生物活性并用作进一步体内修饰的柄。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内。因此，本研究的第一个目标是更精确地定义倍半萜烯羟化酶的结构元件，调节和控制其催化活性。这是通过将一种倍半萜烯羟化酶的催化特异性转化为一种密切相关的羟化酶的催化特异性来实现的，所述转化基于基于两种酶之间的结构和分子比较的组合而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，如抗虫和抗病性，以及基于植物中萜烯代谢的操纵的植物中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的萜烯羟化酶表达的策略，并评估这些植物新的生物活性倍半萜的生物合成，额外的工作将进一步推动倍半萜代谢基因工程的最新进展。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大的跨学科努力，涉及本科生和研究生，博士后助理，特别是来自美国人口的科学代表不足的人，在努力提供新的见解，如何代谢途径可能被设计为使用新兴技术提高价值。

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.