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Unraveling the Catalytic Specificity of Terpene Hydroxylases and Engineering Sesquiterpene Hydroxylation in Plants
Department of Plant and Soil Sciences
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.
2011 Project Description
Our long-term aims are to determine the contributions of sesquiterpenes to plant growth and development and to apply this knowledge towards the development of plants producing novel sesquiterpenoids of importance for agricultural, medical, and other industrial applications. Our approach is to understand sesquiterpene biosynthesis at all levels, from the transcriptional regulation of the genes coding of these biosynthetic capacities to the identification of the residues and peptide regions that control the biosynthetic specificity of these enzymes.
During the past year, we were specifically focused on the molecular dissection of sesquiterpene hydroyxlases. For this purpose we focused on two particular cytochrome P450 enzymes, epi-aristolochene hydroxylase (EAH) and premnaspirodiene oxygenase from Hyoscyamus muticus (HPO). EAH catalyzes the successive hydroxylation of 5-epi-aristolochene, first at the C1 position followed by the second hydroxylation at the C3 position generating capsidiol. In contrast, HPO catalyzes the successive hydroxylation at the C4 position of premnaspirodiene to yield the ketone solavetivone. Both capsidiol and solavetivone possess antimicrobial activities and, because their production in planta is pathogen inducible, they are considered to be phytoalexins.
EAH and HPO are 81% identical at the level of their amino acid sequence comparison and we supposed one means of defining their catalytic specificities would be to interconvert one into the other. For instance, what amino acids of HPO need to be mutated to convert its catalytic specificity to that of EAH. More specifically, could we make reciprocal mutants in HPO, change particular amino acids to those found in EAH, and observe a change in HPO to an EAH like enzyme.
This report updates progress in this effort and details progress in defining the amino acid residues within the EAH enzyme important for the regio-selectivity and successive hydroxylations of 5-epi-aristolochene.
During the last year, we have focused on one particular effort. We continued performing reciprocal mutagenesis of HPO to convert it to an EAH-like enzyme. For this reason, we created mutations in many of the analogous sites as for the HPO to EAH conversion work described above, as well as sites identified by examining molecular models of the EAH and HPO enzymes.
By means of making changes for specific amino acids within the HPO enzyme, we have identified 5 amino acid positions and residues sufficient to account for the successive hydroxylation of 5-epi-aristolochene, first at C1 with beta-orientation and then at C3 with alpha-orientation. The positions and mutants within HPO defined in this manner are I294V (replacing isoleucine at position 294 with valine), F296V, V366S, V482I and A484I. The M5 HPO mutant (the enzyme having all 5 amino acid changes) resulted in a bi-functional enzyme that recapitulated the catalytic specificity found within the wild type EAH enzyme.
This new mutant enzyme exhibited reaction product specificity identical to the wild type EAH, yielding both 1-beta-hydroxy aristolochene and capsidiol upon incubation with 5-epi-aristolochene. Yet, this new mutant enzyme did not capture the quantitative yields of the wild type EAH enzyme. While the EAH enzyme yielded reaction products of approximately 75% capsidiol and 25% 1-beta-epi-aristolochene, the mutant enzyme generated an inverse ratio of products, approximately 20% capsidiol and 80% 1-beta-hydroxy epi-aristolochene.
Various mutants were also examined for their ability to directly utilize the 1-beta-hydroxy-5-epi-aristolochene substrate. While the V366S, V482I, A484I mutant is unable of using the mono-hydroxylated substrate, addition of the 294/296 mutations to the M3 HPO mutant provided a new gain of function activity. The M5 mutant readily accepted 1-beta-hydroxy-5-epi-aristolochene as a substrate, and equally important, yielded a single hydroxylated product, capsidiol, much like the wild type EAH enzyme.
Albeit the M5 mutant exhibited only about 20% of the turnover rate of EAH enzyme for the mono-hydroxylated substrate, the combination of these 5 residues appear sufficient to account for the regio- and successive hydroxylation specificity found in the EAH enzyme.