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

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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.