Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: 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 metabolism in Valeriana officinalis for a three reasons. One, V. officinalis makes an unusual series of sesquiterpenes associated with the nutraceutical value of V. officinalis. V. officinalis root extracts are sold as a natural source for the treatment of sleeplessness and anxiety. Second, the putative active ingredient sesquiterpenes are unusual chemical structures for which there wasn't any known mechanism for their biosynthesis. Third, the biological sesquiterpenes of V. officinalis have regio-specific hydroxylations. It is our contention that by being able to compare the enzymes of V. officinalis responsible for hydroxylation with those for responsible for sesquiterpene hydroxylation in other plant species, should help use understand the molecular wizardry of these enzymes. The current report, hence, tracks progress in determining the biosynthetic pathway for valerianadiene biosynthesis. PARTICIPANTS: Joe Chappell, PI, University of Kentucky, Yun-Soo Yeo, Postdoctoral Assoc., University of Kentucky, Eric Nybo, Postdoctoral Assoc., Scott Kinison, Lab Tech, University of Kentucky TARGET AUDIENCES: Science students at multiple levels - secondary school through graduate students targeted in our DNA Science Workshops offered regionally and by formal classes offered at the University of Kentucky. Practicing scientists at public and private national and international institutions reached by participation at scientific congresses and giving seminars at UK public institutions. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Valerian is an herbal preparation from the roots of Valeriana officinalis used as an anxiolytic and sedative, and in the treatment of insomnia. The biological activities of valerian are attributed to valerenic acid and its putative biosynthetic precursor valerenadiene, sesquiterpenes found in V. officinalis roots. These sesquiterpenes retain an isobutenyl side chain whose origin has been long recognized as enigmatic because a chemical rationalization for their biosynthesis has not been obvious. Using recently developed metabolomic and transcriptomic resources, we identified seven V. officinalis terpene synthase genes (VoTPSs), two that were functionally characterized as monoterpene synthases and three that preferred farnesyl diphosphate (FPP), the substrate for sesquiterpene synthases. The reaction products for two of the sesquiterpene synthases exhibiting root specific expression were characterized by a combination of GC-MS and NMR in comparison to the terpenes accumulating in planta. VoTPS7 encodes for a synthase that biosynthesizes predominately germacrene C, while VoTPS1 catalyzes the conversion of FPP to valerena-1,10-diene. Using a yeast expression system, specific labeled 13C acetate and NMR, we investigated the catalytic mechanism for VoTPS1 and provide evidence for the involvement of a caryophyllenyl carbocation, a cyclobutyl intermediate, in the biosynthesis of valerena-1,10-diene. We suggest a similar mechanism for the biosynthesis of several other biologically related isobutenyl containing sesquiterpenes.
Publications
- Yeo, Y.S., Nybo, S.E., Chittiboyina, A.G., Weerasooriya, A.D., Wang, Y.H., Gongora-Castillo, E., Vaillancourt, B., Buell, C.R., Dellapenna, D., Celiz, M.D., Jones, A.D., Wurtele, E.S., Ransom, N., Dudareva, N., Shaaban, K.A., Tibrewal, N., Chandra, S., Smillie, T., Khan, I.A., Coates, R.M., Watt, D.S., Chappell, J. (2012) Functional identification of valerena-1,10-diene synthase, a terpene synthase catalyzing a unique chemical cascade in the biosynthesis of biologically active sesquiterpenes in Valeriana officinalis. Journal of Biological Chemistry DOI 10.1074/jbc.M112.415836
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: 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. PARTICIPANTS: Joe Chappell, PI, University of Kentucky Jeanne Rasbery, Postdoctoral Assoc., University of Kentucky Keith Allen, Graduate Student, University of Kentucky Scott Kinison, Lab Tech, University of Kentucky worked on this project during the last year. TARGET AUDIENCES: Science students at multiple levels - secondary school through graduate students targeted in our DNA Science Workshops offered regionally and by formal classes offered at the University of Kentucky (ABT 495). Practicing scientists at public and private national and international institutions reached by participation at scientific congresses and giving seminars at UK public institutions. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
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.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: 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. As one approach toward achieving these goals, we aim to understand sesquiterpene biosynthesis by identifying the residues and peptide regions that control sesquiterpene hydroxylase specificity. And for this purpose we have focused our attention to two particular cytochrome P450 enzymes, epi-aristolochene hydroxylase (EAH) and premnaspirodiene oxygenase from Hyoscyamus muticus (HPO). Both hydroxylases rely on native sesquiterpene scaffolds generated by terpene synthases. 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? We succeeded in this endeavor and made the following key observations. Combined mutations V366S (valine to serine mutation at position 366 of HPO), V482I and A484I were sufficient to change the regio-specificity of the initial hydroxylation from C2 of aristolochene to C1. Further mutations of I294V and F296V improved overall catalytic efficiency at C1 about 2-fold. However, no successive hydroxylation at C3 was observed in these in vitro reactions. Additional mutations V109I and H238L did however uncover the capacity of the mutant HPO to synthesize capsidiol in addition to 1β-hydroxyl aristolochene. The mutant HPO activity however was not an exact replication of authentic EAH activity. Although the mutant HPO was about 2 times more active than EAH, it only generated about 10% of its reaction product as capsidiol while the wild type EAH generates 75-80% of its reaction products as capsidiol. PARTICIPANTS: Joe Chappell, PI, University of Kentucky Jeanne Rasbery, Postdoctoral Assoc., University of Kentucky Keith Allen, Graduate Student, University of Kentucky Scott Kinison, Lab Tech, University of Kentucky TARGET AUDIENCES: Science students at multiple levels - secondary school through graduate students targeted in our DNA Science Workshops offered regionally and by formal classes offered at the University of Kentucky (ABT 495). Practicing scientists at public and private national and international institutions reached by participation at scientific congresses and giving seminars at UK public institutions. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
During the last year, we have focused on 2 efforts. The first was to perform the reciprocal mutagenesis of EAH to convert it to an HPO-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. Analogous to position F296 in HPO, V298 when changed to phenylalanine (F) in EAH greatly enhanced overall catalytic activity, as did mutants S368V, I484V and S368V, I486A. In the earlier mutagenesis work with HPO, positions analogous to 368, 484 and 486 were more associated with the regio-specificity of the enzyme rather than overall catalytic activity. When additional residues at positions 52, 107, 109, and 209 of EAH were mutated to the corresponding residues found in HPO, the mutant enzymes appeared to take on more of the successive nature of catalysis and specifically for generation of the ketone product solavetivone. These particular mutants were selected because of the prior work with the corresponding positions in HPO and because of their putative position within the molecular models of EAH. Confirmation of a role for these residues in the successive hydroxylation activity of the EAH mutant was provided by examining their catalytic activity against alternative substrates. For instance, mutants D107E; I109V, E209G; E52L, E209G; or E209G in combination with S368V and S482V all exhibited significant successive hydroxylation of 1β-hydroxy-epi-aristolochene to its ketone form. Our second major effort over the last year has been focused on the isolation of yet other sesquiterpene hydroxylases that exhibit regio-specificities different than EAH or HPO, and in particular those associated with parthenolide biosynthesis. It is our hope that the identification of hydroxylases which hydroxylate at different positions around the sesquiterpene scaffolds can be used in additional work to further improve our understanding of what structural features of these enzymes control regio-specificity. And if that were possible, then perhaps we would be able to predictably create hydroxylases via mutagenesis that decorated sesquiterpene scaffolds at particular carbon positions, and thus create novel molecules. Our efforts have focused on the hydroxylases associated with parthenolide biosynthesis Magnolia grandiflora. RNA was isolated from young developing leaves and subject to 454 pyrosequencing. Over 270,000 reads yielded approximately 58 million bp with an average read of 215 bp. About one-third of the sequences assembled into contigs, which were depositing into a NCBI blast server platform. The sequence information was then screened for possible hydroxylase like genes using the EAH and HPO gene sequences, along with several other well described terpene hydroxylating enzymes. Three candidate genes have been identified and are slated for functional screening of substrate and reaction product specificity after expression in our yeast expression system.
Publications
- Faraldos, J.A., Wu, S., Chappell, J. and Coates R.M. (2010) Doubly Deuterium-Labeled Patchouli Alcohol from Cyclization of Singly Labeled [2-(2)H(1)]Farnesyl Diphosphate Catalyzed by Recombinant Patchoulol Synthase. J Am Chem Soc. 132:2998-3008
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 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. PARTICIPANTS: Joe Chappell, PI, University of Kentucky Jeanne Rasbery, Postdoctoral Assoc., University of Kentucky Shuiqin Wu, Postdoctoral Assoc., University of Kentucky Scott Kinison, Lab Tech, University of Kentucky TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
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.
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.
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