By Shannadora Hollis
The medicinally important sesquiterpenes of Valeriana officinalis are biosynthesized in their roots. Courtesy of the National Center for Natural Products Research at the University of Mississippi.
While plant products continue to be a prime target for drug development, their clinical potential is often curtailed by scientists’ inability to identify and synthesize sufficient and consistent levels of the plant’s bioactive components. This has proved to be the case for the plant Valeriana officinalis, commonly known as valerian.
For a recent study published in The Journal of Biological Chemistry, researchers at the University of Kentucky set out to examine the biosynthesis of valerian in hopes of elucidating where valerenadiene, the plant’s proposed bioactive component, is expressed and how it is synthesized.
Historical and biochemical origins of valerian
Valeriana officinalis is a perennial native to Europe and Asia. It was used as a medicinal herb in ancient Greece and Rome for the treatment of insomnia, with its therapeutic uses described by the likes of Hippocrates and Galen (1, 2).
This tradition continues today. Herbal preparations of valerian still are used as a remedy for insomnia as well as anxiety. The biological activities of the plant are attributed to valeranone, valerenadiene and valerenic acid (3). These compounds are typically found in the plant roots and are derived from a larger class of chemicals known as sesquiterpenes. Valeranone may be derived from germacrene, while the biosynthetic precursor for valerenic acid is thought to be valerena-1,10-diene, also known as valerenadiene.
Valerian’s chemical structure: unraveling the sesquiterpene isobutanyl side chain
According to Joe Chappell, professor of plant biochemistry at the University of Kentucky and director of the Medicinal Plant Consortium, “Chemicals like you find in Valeriana, especially the sesquiterpene molecules, have represented a conceptual challenge ... because they’re very unique in their structure.”
More specifically, sesquiterpenes contain an isobutenyl side chain. The origin of this evolutionarily conserved functional group has been enigmatic, as a chemical rationalization for its biosynthesis has not been readily apparent. “Chemists have pondered how these molecules can be made biosynthetically for well over 100 years,” adds Chappell.
To understand how the biosynthesis of such chemicals and their respective substituent groups occurs, chemists often start from the beginning of the process: by looking at the enzymes that catalyze the biochemical reactions of interest. “One would have to go in and study the enzymes themselves to really understand how you get this unique isobutenyl side chain,” Chappell says.
This fundamental chemistry principle led researchers to attempt to identify the enzymes that catalyze the biosynthesis of valerena-1,10-diene, the structurally elusive isobutenyl side chain-bearing sesquiterpene.
Identifying valerian’s catalytic enzyme: reaching into the bioinformatic toolbox
“Plants harbor an unbelievable array of molecules. Many of these molecules are made by enzymatic catalysts that use unprecedented chemical transformations,” says Sarah O’Connor, co-director of the Medicinal Plant Consortium. “With the increasing availability of plant sequence data, along with the bioinformatic tools to mine it, we can find these enzymes much more rapidly.”
This was precisely the approach employed by Chappell et al. in the valerian study. The investigators first used the Medicinal Plant Genomics Resource and Medicinal Plant Metabolomics Resource. These searchable and downloadable online databases were made available to the research community in 2011 as part of an ongoing initiative to study how plant genes contribute to the production of various chemical compounds and to assist in the advancement of plant-derived drug development.
The project, led by Chappell, was part of a $6 million collaborative Grand Opportunities grant through the National Institutes of Health and the National Institute of General Medical Sciences. Researchers from seven institutions studied the genes and chemical profiles of 14 plants known for medicinal properties, including Valeriana officinalis. More specifically, they determined the DNA sequences and expression profiles of the transcriptomes and the associated metabolomes of the plants, thereby allowing identification of candidate pathway genes.
The researchers took advantage of these bioinformatic tools to study Valeriana officinalis, using the databases to screen the various valerian tissue types (leaves, flowers, roots, etc.) for metabolites that yielded fragment ions with a mass-to-charge ratio characteristic of a derivative of valerena-1,10-diene.
Several ions meeting this criterion were found exclusively in the root tissues. One corresponded to an acetylated sesquiterpenoid diol. Sesquiterpenoids are biochemically modified sesquiterpenes. Their formation is catalyzed by the enzyme terpene synthase. Thus, the presence of this modified sesquiterpene in the root of valerian suggested that the terpene synthase(s) responsible for the biosynthesis of valerenadiene also might be in the root.
Chappell and his team identified seven possible Valeriana officinalis terpene synthase (VoTPS) gene candidates that may be involved in the synthesis of valerena-1,10-diene. To biochemically characterize these putative genes, cell-free extracts from E. coli expressing each of the VoTPS cDNAs encoding for the respective genes were expressed and analyzed to determine their enzymatic activity and reaction products.
Using gas chromatography-mass spectrometry and nuclear magnetic resonance techniques, the team determined that the reaction product of one of the candidate terpene synthase genes, VoTPS1, was valerenadiene. This confirmed that VoTPS1 was indeed the enzyme responsible for the catalysis of the sesquiterpene.
Revisiting “paper-pencil chemistry” to fill in the mechanistic gaps
With the enzyme responsible for the synthesis of valerena-1,10-diene identified, researchers next relied on the biosynthetic logic of their scientific predecessors to shed light on the origin of the sesquiterpene’s enigmatic isobutenyl side chain. Joseph D. Connolly, a chemist at the University of Glasgow, previously addressed this mechanistic uncertainty by suggesting the possible involvement of a caryophyllene intermediate. Caryophyllene is a natural bicyclic sesquiterpene that is notable for having a cyclobutane ring — a rarity in nature.
“Dr. Connolly was one of the theoretical chemists that were thinking about how you get this isobutenyl group,” Chappell explains. “He proposed the intermediate as a way to get the four-carbon substituent group generated. We took his suggestion of the caryophyllene intermediate…played pretty hard with it in paper-pencil chemistry, and we ultimately came up with a proposal for how the chemical transformation occurred…Then we wanted to test it by looking at 13C incorporation into valerenadiene. We could make a prediction about what carbons would be adjacent to one another in the molecule using NMR techniques.”
The team observed a mechanism consistent with the expected labeling pattern of the proposed caryophyllene reaction mechanism for valerena-1,10-diene catalysis by VoTPS1. In this mechanism, C1 forms a bond with C11 of farnesyl diphosphate, the substrate for terpene synthases. This results in the formation of a caryophyllenyl carbocation, followed by opening of the cyclobutyl ring to yield the desired isobutenyl side chain.
Is it time to put the enigma to bed?
Medicinal plants produce a wealth of pharmaceutical compounds. Unfortunately, the metabolic pathways involved in the synthesis of these compounds remain poorly understood. Elucidating these pathways has been difficult in part due to the limited availability of genetic resources for these plants.
Thanks to state-of-the-art, high-throughput genomic and transcriptomic sequencing resources and bioinformatic tools, research in the life sciences has experienced what many would consider a revolution. For the first time, these advances have been extended to the medicinal plant arena on a broad scale, and their influence is prominent in this study on Valeriana officinalis.
According to Fabricio Medina-Bolivar, professor of plant metabolic engineering and vice president of the American Council for Medicinally Active Plants, “This work opens the doors to study the biosynthesis of related sesquiterpenes and highlights the importance of newly available transcriptomic data to identify key enzymes involved in the biosynthesis of medicinally active compounds.”
Valerian has created quite a buzz in the scientific community, as evidenced by numerous clinical trials involving the plant (4, 5, 6, 7, 8, 9). Although the biological activities of valerian are attributed to sesquiterpenes, whether these compounds are responsible for the reported sedative effects of the plant “remains to be seen,” says Chappell.
“We have now provided the research community with a means for producing this compound [valerenadiene] by itself in high yields and with a high purity,” he says. “Now there will be lots of follow-up testing to determine if this is really the molecule that is providing the anti-anxiety and sleep induction benefits.”
REFERENCES
1. Blumenthal, M., Goldberg, A., & Brinckmann, J., eds.: “Valerian root.” Herbal Medicine: Expanded Commission E Monographs. (2000).
2. Turner, W. “Of Valerianae.” A New Herbal, Parts II and III. (1995). [Republication of parts II and III of A New Herbal by William Turner, originally published in 1562 and 1568, respectively.]
3. Takemoto, H., et al. J. Natural Medicines 63, 380 – 385 (2009).
4. Stevinson, C. & Ernst, E. Sleep Medicine 1, 91 – 99 (2000).
5. Jadad, A.R. et al. Controlled Clinical Trials 17, 1 – 12 (1996).
6. Leathwood, P.D. et al. Pharmacology, Biochemistry and Behavior 17, 65 – 71 (1982).
7. Leathwood, P.D. & Chauffard, F. Planta Medica 2, 144 – 148 (1985).
8. Dorn, M. Forschende Komplementärmedizin und Klassische Naturheilkunde 7, 79 – 84 (2000).
9. Donath, F. et al. Pharmacopsychiatry 33, 47 – 53 (2000).
Shannadora Hollis (sholl002@umaryland.edu) received her B.S. in chemical engineering from North Carolina State University and is a Ph.D. student in the molecular medicine program at the University of Maryland, Baltimore. Her research focuses on the molecular mechanisms that control salt balance and blood pressure in health and disease. She is a native of Washington, D.C., and in her spare time enjoys cooking, thrift-store shopping and painting.
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