Toni Kutchan

Prior to joining the Danforth Center in 2006, Dr. Kutchan spent 20 years researching biochemistry at the University of Munich and the Leibniz Institute of Plant Biochemistry in Germany. She is currently investigating how plants produce medicinal compounds at the enzyme and gene level, which could lead to new sources of medications for use against conditions such as chronic pain and cancer. Dr. Kutchan serves as Adjunct Professor of Biology at Washington University. She received her B.S. in Chemistry at the Illinois Institute of Technology and her Ph.D. in Biochemistry at St. Louis University.

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Research

Our laboratory research aims at elucidating the biosynthetic pathways of selected medicinal compounds in plants and developing improved sources of these chemicals.

We investigate how plants make special chemicals called natural products. These chemicals frequently are used as medicines, either as pure compounds by pharmaceutical industry, or as mixtures in traditional medicines. Selected natural products are currently being investigated in the laboratory in mature plants and in tissue and cell culture. We participate in three national and international projects that involve deep transcriptome sequencing of medicinal plants using next generation sequencing technologies. In general, in our research, we strive to understand the formation of medicinal compounds in selected plants at the enzyme and gene levels and then to use this information to improve upon production of pharmaceuticals either in planta or in a heterologous host such as yeast or bacteria.


Evolution of pathways to pharmaceuticals in the poppy family

To date, only partial understanding of the formation of medicinal alkaloids at the enzyme and gene levels has been attained. In addition, the evolutionary origins of these biosynthetic pathways remain unsolved. The explosive increase in understanding of biology over the past two decades has been enabled by work on model genetic organisms, including the plant Arabidopsis thaliana. The study of selected species-specific medicinal secondary metabolites, however, requires investigation of those plant species that harbor all or most components of the focal biosynthetic pathway. Detailed genetic and biochemical information on these highly specialized species is often missing. This knowledge gap slows research advances in the field of plant-derived pharmaceuticals. The long-term goal of this research is to understand the complete formation and evolutionary origins of isoquinoline alkaloids such as morphine, papaverine and sanguinarine at the enzyme and gene level. Improved understanding will enable the development of alternate sources of alkaloids that lie along the biosynthetic pathway and to develop novel drugs. The objectives of this project are i) to execute cross-species comparative analyses of the enzyme-coding and regulatory genes involved in alkaloid biosynthesis, ii) to elucidate the evolutionary origins of pathways producing specific isoquinoline alkaloids, and iii) to identify unknown genes/enzymes in isoquinoline alkaloid biosynthetic pathways. The specific aims are 1) Associate variation in gene expression with variation in alkaloid profiles in poppy species, 2) Reconstruct the evolution of alkaloid biosynthetic pathways in the poppy family and determine how gene duplications and adaptive amino acid substitutions have resulted in metabolite diversification, 3) Preliminary functional analysis of selected poppy genes predicted to encode enzymes involved in the biosynthesis of medicinal alkaloids. The resultant database and tools for comparative analysis will lead to gene discovery and enable technologies in the broader medicinal plant field, thereby positively impacting the development of novel drugs and the production of known drugs. It is expected that the entire field of plant-derived pharmaceuticals will be advanced to a higher, more comprehensive level of analysis as a result of our research.


Transcriptome characterization of medicinal plants relevant to human health

Plants are the source of many important medicinal compounds and the diversity of plant species and biochemistry suggests that many more are potentially available. The current understanding of the formation of plant-derived medicinal compounds at the enzyme, gene and regulatory levels is very incomplete—not a single complex plant medicinal pathway has yet to be completely elucidated at both the enzyme and the regulatory level. Historically, most studies of plant-derived medicinal compounds have been very narrowly focused, typically devoted to very specific steps in a particular biosynthetic pathway. These investigations were often pioneering—the diversity of plant biochemistry contains many novel reactions—but they were also very labor-intensive. More recently, genome-wide studies of model plant species have resulted in an explosive increase in our knowledge of, and capacity to understand, basic biological processes. Working from the genetics to the biochemistry now provides the most efficient way to build a long awaited and urgently needed foundation for more effectively probing and exploiting plant medicinal compound biosynthetic pathways. Having a comprehensive medicinal plant transcriptome database would propel medicinal plant species from an orphan-like status into the limelight of plant biochemistry and molecular genetics. The long-term goal of this research is for the scientific community to understand the complete formation, storage and regulation of plant-derived medicinal compounds at the enzyme and gene level. Improved understanding will enable the development of alternate sources of known pharmaceuticals and of novel drugs. The objective of this project is to provide the research community with urgently needed infrastructure and resources to enable comprehensive studies of the most compelling and medicinally significant plant biosynthetic pathways. Our Specific Aims are 1) To validate twenty selected medicinal plants based on taxonomic classification, medicinal compound accumulation and target transcript analysis, 2) To conduct transcriptome profiling of these medicinal plants and 3) To disseminate the accumulated data to the scientific community in the form of a user-friendly database. The resultant database and tools for comparative analysis will lead to gene discovery and enable technologies in the broader medicinal plant field. It is expected that the entire field of plant-derived pharmaceuticals will be advanced to a higher, more comprehensive level of analysis as a result of the proposed research. This is significant because the results will provide researchers in the field of plant-derived pharmaceuticals with a publicly available database and search tools for comparative analyses of pathway enzyme-coding genes and regulatory factors. These tools will enable gene discovery, metabolic engineering, synthetic biology and directed evolution for the improved production of drugs and for the development of novel drugs. Taken together, we envisage that this will ultimately result in tangible long-term and meaningful benefits for public health.


1000 Plant Transcriptome

This Canadian project funded by the government of Alberta proposes to sequence and assemble 1000 de novo plant transcriptomes using Illumina sequencing technology. The assembled sequences will be made publicly available. It will be initially sought to greatly increase the number of plant species for which transcript sequence information is publicly available and to learn about the biology of these plants and evolutionary history. Our role in this project focuses mainly on the poppy family and complements the two projects described above.


Opium Poppy Papaver somniferum

The opium poppy Papaver somniferum is one of mankind’s oldest medicinal plants and serves today as the commercial source of the powerful analgesic morphine, from which a variety of analgesics and antitussive alkaloids, such as codeine, are derived by semi-synthesis. The morphine biosynthetic intermediate thebaine is a synthetic starting material for the production of the analgesics oxycodone and oxymorphone, as well as for the synthesis of potent opiate antagonists such as naloxone and naltrexone.
Although formal syntheses of morphine have been reported, the morphine moleculecontains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P-450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P-450 during a screen of recombinant cytochromes P-450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo- and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P-450 that catalyze formation of a phenol couple.

The Figure above shows the atomic structure of the morphine biosynthetic enzyme salutaridine reductase bound to the cofactor NADPH. The substrate salutaridine is shown entering the active site. (structure by Tom Smith; photo by R.H. Berg)

Our past research suggests that there is a physical interaction between at least the two enzymes of morphine biosynthesis salutardine reductase and salutaridinol acetyltransferase. To further pursue our study of protein-protein interactions in the morphine biosynthetic pathway, we have crystallized salutardine reductase and have solved the crystal structure with the cofactor NADPH together with Dr. Tom Smith of the Donald Danforth Center Plant Science Center. This is the first enzyme of morphine biosynthesis for which a crystal structure has been determined.

The Figure above shows crystals of Papaver somniferum SalR grown under different conditions.


New publications related to our opium poppy research

Kempe, K., Higashi, Y., Frick, S., Sabarna, K. and Kutchan, T.M. RNAi suppression of the morphine biosynthetic gene salAT and evidence of association of pathway enzymes. Phytochemistry, 70, 579-589 (2009).

Gesell, A., Rolf, M., Díaz Chávez, M.L., Huang, F.-C., Ziegler, J. and Kutchan, T.M. CYP719B1 is salutaridine synthase, the phenol-coupling enzyme of morphine biosynthesis in opium poppy. J. Biol. Chem. 284, 24432-24442 (2009).

Higashi, Y., Smith, T.J., Jez, J.M. and Kutchan, T.M. Crystallization and preliminary X-ray diffraction analysis of salutaridine reductase from Papaver somniferum. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. F66, 163-166 (2010).

Higashi, Y, Kutchan, T.M., and Smith, T.J. The atomic structure of salutaridine reductase from the opium poppy Papaver somniferum. J. Biol. Chem. (2010) doi:10.1074/ jbc.M110.168633.

Gesell, A., Díaz Chávez, M.L., Kramell, R., Piotrowski, M., Macheroux, P., and Kutchan, T.M. (2011). Heterologous expression of two FAD-dependent oxydases with (S)-tetrahydroprotoberberine oxidase activity from Argemone mexicana and Berberis wilsoniae in insect cells. Planta, doi:10.1007/s00425-011-1357-00424.

Ipecac Psychotria ipecacuanha

The medicinal plant Psychotria ipecacuanha produces Ipecac alkaloids, a series of monoterpenoid-isoquinoline alkaloids such as emetine and cephaeline, whose biosynthesis derives from condensation of dopamine and secologanin. We have identified three cDNAs, IpeOMT1-IpeOMT3, encoding Ipecac alkaloid O-methyltransferases (OMTs) from P. ipecacuanha. They were coordinately transcribed with our recently identified Ipecac alkaloid β-glucosidase Ipeglu1. The amino acid sequences were closely related to each other, and rather to the flavonoid OMTs than to the OMTs involved in benzylisoquinoline alkaloid (morphine) biosynthesis. Characterization of the recombinant IpeOMT enzymes with integration of the enzymatic properties of the IpeGlu1 revealed that emetine biosynthesis branches off from N-deacetylisoipecoside through its 6-O-methylation by IpeOMT1, with a minor contribution by IpeOMT2, followed by deglucosylation by IpeGlu1. The 7-hydroxy group of the isoquinoline skeleton of the aglycon is methylated by IpeOMT3 prior to the formation of protoemetine that is condensed with a second dopamine molecule, followed by sequential O-methylations by IpeOMT2 and IpeOMT1 to form cephaeline and emetine, respectively. In addition to this central pathway of Ipecac alkaloid biosynthesis, formation of all methyl derivatives of Ipecac alkaloids in P. ipecacuanha could be explained by the enzymatic activities of IpeOMT1-IpeOMT3, indicating that they are sufficient for all O-methylation reactions of Ipecac alkaloid biosynthesis.


New publication related to our Ipecac alkaloid research

Nomura, T. and Kutchan, T.M. Three new O-methyltransferases are sufficient for all O- methylation reactions of Ipecac alkaloid biosynthesis in root culture of Psychotria ipecacuanha. J. Biol. Chem.285, 7722-7738 (2010) doi:10.1074/jbc.M109.086157.

Nomura, T. and Kutchan, T.M. Is a metabolic enzyme complex involved in the efficient and accurate control of ipecac alkaloid biosynthesis in Psychotria ipecacuanha? Plant Signal. Behav. 5, 875-877 (2010).


Morphine in mammals

Morphine, one of the strongest analgesic compounds known in human physiology is administered by ingestion or injection. It is a major alkaloid in the latex of the plant Papaver somniferum. Morphine has been found to be present in trace amounts in human cells and in ca. 10 nM concentration in mammals and the question was – is it of dietary origin or does it occur endogenously? Studies done in our lab have shown for the first time that morphine is biosynthesized in mammalian tissues such as human neuroblastoma- and pancreas carcinoma cells. Incorporation experiments with ¹⁸O₂ and feeding experiments involving heavy isotope-labeled precursors like DOPA have shown unequivocally that morphine found in mammals is of endogenous and not of dietary origin. With these results we developed a putative pathway for the biosynthesis of endogenous morphine in mammals and compared this pathway with that in the poppy plant. Enzymatic studies revealed first clues as to the enzymatic mechanisms involved in the formation of endogenous morphine in mammals and confirmed the proposed biosynthetic pathway. Our main interest focuses presently on the discovery of enzymes in mammals being involved in the biosynthesis of endogenous morphine and on the detection of endogenous morphine in mammalian tissue, especially brain.

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R) reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H. and Zenk, M. H. (1995) Heterocycles 40, 425-440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (-)-isoboldine, (-)-corytuberine, (+) pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (-)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b₅ with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nM concentration of morphine found in human blastoma cell cultures and in brain tissues of mice.


New publication related to our mammalian morphine research

Grobe, N., Lamshöft, M., Orth, R.G., Dräger, B., Kutchan, T.M., Zenk, M.H. and Spiteller, M. Urinary excretion of morphine and biosynthetic precursors in mice. Proc. Natl. Acad. Sci. USA 107, 8147-8152 (2010).

Han, X., Lamshöft, M., Grobe, N., Ren, X., Fist, A.J., Kutchan, T.M., Spiteller, M. and Zenk, M.H. The biosynthesis of papaverine proceeds via (S)-reticuline. Phytochemistry 71, 1305-1312 (2010).

Grobe, N., Ren, X., Kutchan, T.M. and Zenk, M.H. An (R)-specific N-methyltransferase involved in human morphine biosynthesis. Arch. Biochem. Biophys. 506, 42-47 (2011).

Frölich, N., Dees, C., Paetz, C., Ren, X., Lohse, M.J., Nikolaev, V.O., and Zenk, M.H. (2011). Distinct pharmacological properties of morphine metabolites at G(i)-protein and β-arrestin signaling pathways activated by the human μ-opioid receptor. Biochem. Pharmacol., doi:10.1016/j.bcp.2011.03.001.


Center for Advanced Biofuel Systems (CABS)

Jet fuel is a mixture of many different hydrocarbons. Modern analytical techniques indicate that there may be a thousand or more. The range of their sizes (carbon numbers) is restricted by specific physical requirements of a specific jet fuel product. Kerosine-type jet fuel has a carbon number distribution between about 8 and 16 carbons. Most of the hydrocarbons in jet fuel are members of the paraffin, naphthene and aromatic classes. The compounds that boil near the middle of the kerosine-type jet fuel boiling-range are C10 aromatics, C11 naphthenes, and C12 waxes. Plants synthesize a wide repertoire of cyclic and linear low molecular weight compounds. An introduction of relatively few low molecular weight metabolite biosynthetic genes into a heterologous host such as an oilseed or an alga could result in the production and accumulation of low molecular weight hydrocarbons that could serve as chemical precursors to aromatics, naphthenes or waxes. In planta, C-10 terpenes (monoterpenes) are synthesized in plastids of specialized gland cells from precursors derived via the non-mevalonate pathway from pyruvate and glyceraldehyde-3-phosphate. C-15 terpenes (sesquiterpenes) are synthesized in the cytosol via the mevalonate pathway from acetyl-CoA. The volatile products of mono- and sesquiterpene biosynthesis are either secreted into specialized storage cavities or are released to the atmosphere. We are attempting to biosynthesize and accumulate mono- and sesquiterpenes in plastids and cytosol in oilseed and algae towards the ultimate

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