Research Accomplishment Reports 2009

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

J. Chappell
Department of Plant and Soil Sciences

 

Non-Technical Summary

Terpenes, which represent a complex array of chemical compounds that are essential for many aspects of plant growth and development, are of considerable importance for their nutritional contributions to animals and humans, 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 biosynthesis has been intensively studied. Nonetheless, our appreciation for many of the mechanistic features of these enzymes involved in these biosynthetic pathways is still very limited.

The current application 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 or several hydroxyl substituents in very specific patterns that impart biological activities to the sesquiterpene products 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 application is to more precisely define the structural elements of sesquiterpene hydroxylases that regulate and control their catalytic activities. We propose to accomplish this by interconverting 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. Ultimately, we aim to create new plant traits, like enhanced insect and disease resistance, and the biosynthesis of high-value natural products in plants based upon the manipulation of terpene metabolism in plants.

Our second objective, therefore, will further recent advances in the genetic engineering of 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.

2009 Project Description

The long-term goals of this project are to determine the contributions of sesquiterpenes to plant growth and development and to apply this knowledge toward engineering plants that have increased production of sesquiterpenoids important for agricultural, medical, and other industrial applications. To achieve these goals, we sought to further understand sesquiterpene biosynthesis by identifying the residues and peptide regions that control sesquiterpene hydroxylase specificity. In addition, we want to determine if sesquiterpene hydroxylases can be engineered in plants to produce hydroxylated sesquiterpene compounds of agricultural, industrial and medicinal interest.

Our work has been disseminated in presentations at other universities (University of Tottori (Tottoria, Japan), University of Missouri-Columbia, and Southern Illinois University) and at professional societal meetings (Terpnet 2009, Gordon Research Conference, Internal Society of Plant Molecular Biology), as well as by presentations to undergraduate and graduate students at the University of Kentucky.

2009 Impact

To carry out this work, we chose 2 sesquiterpene P450 enzymes of interest, EAH and HPO. These proteins are 81% identical and their activities have been reported. EAH carries out hydroxylation of epi-aristolochene to yield capsidiol whereas HPO carries out hydroxylation of premnaspirodiene to yield the ketone, solavetivone. Capsidiol and solavetivone possess anti-fungal activities and thus are classified as phytoalexins. Using a molecular modeling approach, we identified 25 residues of interest that may function in regulating the regio- and stereo-specificity of the enzymes. Through molecular modeling studies relative to the mammalian 2B4 P450 enzyme, we prioritized 9 residues for mutation and analysis.

Using site-directed mutagenesis, we converted HPO into an enzyme producing capsidiol. Using these data, we postulated that if these residues influence hydroxylation specificity, the reciprocal mutations in EAH should yield an enzyme capable of producing solavetivone with similar HPO activity. The work of the past year has been focused on mutagenesis of EAH. To validate our hypothesis, we created the reciprocal mutations in EAH with the hopes of converting EAH into an HPO-like enzyme. Single, double, triple, and quadruple mutations have been created in EAH and activity has been evaluated by in vitro analysis.

For a better understanding of how these residues influence enzyme activity, we evaluated select EAH mutant activities when given EA (5-epi-aristolochene) or premnaspirodiene, as substrates. Mutations at positions 368, 482, and 486 were of particular interest due to their apparent projection into the active site and due to high residue variability among P450 family members at these positions. The S368V I486A double mutant appears to have reduced enzymatic activity as solavetivol, solavetivone, and capsidiol production appear decreased in this mutant. Introducing an additional mutation in this background (E209G) appears to abolish capsidiol production and results in an enzyme that produces epi-aristolochen-1-one instead. In addition, solavetivone production is abolished and solavetivol production appears similar to that of wild-type EAH. Combining S368V with S482V appears to have very different consequences for EAH activity. Combining this double mutants with D107E results in an enzyme that produces significantly higher levels of 1β(OH)EA, yet markedly lower levels of solavetivol and solavetivone. The quadruple mutant (I109V E209G S368V S482V) results in an enzyme that produces higher levels of 1β(OH)EA and epi-aristolochen-1-one. In addition this mutant produces higher levels of solavetivone than EAH and increased hydroxylation of valencene to yield nootkatol and nootkatone. The quadruple mutant E52L E209G S368V S482V also produces more solavetivone than wild type, but appears to have significantly reduced activity on 5-EA. These data point to a role for S368V and S482V in the hydroxylation of compounds to produce ketone products as we noticed increased production of solavetivone, epi-aristolochen-1-one and nootkatone.

2009 Publications

Chappell, J. and Coates, R. M. (2009) Sesquiterpenes, Chapter 5 in Comprehensive Natural Products Chemistry II, C. Townsend and Y. Ebizuka eds, Elsevier Ltd, London, England, pp. 1-33.