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.
RESEARCH TEAM |
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
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
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
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
Technologies available for license:
Toni Kutchan, Ph.D.Member, Oliver M. Langenberg Distinguished Investigator, VP for Research
975 N. Warson Rd.
St. Louis, MO 63132