A presentation on the O'Connor group, given as part of the Advanced Topics of Chemical Biology module. Yet to be awarded a mark, I presented from slide 6 onwards.
J. Ingram, D. Poulcharidis - Adv. Topics of Chem. Bio. - Dr. Webb - Prof. S. O'Connor Presentation
1. 1
Advanced Topics of Chemical
Biology
- Dr. M. Webb, University of Leeds
J. Ingram, D. Poulcharidis
- Prof. S. O'Connor
2. 2
The O’Connor Group
Investigates how plants and plant pathogens produce complicated molecules
from simple metabolic building blocks.
• Anti-cancer : Vinblastine and Taxol
• Analgesic: Morphine
• Anti-malarial: Artemisinin and Quinine
Catharanthus roseus
vinblastine
Taxol
Taxol
Taxus brevifolia
Claviceps purpurea
Ergotamine Morphine
Papaver somniferum
3. 3
Areas of Interest
• Metabolic Pathway Elucidation in Medicinal Plants.
• Synthetic Biology with Metabolic Pathways.
• Enzyme Biochemistry, Mechanism and Evolution.
A major focus in the group is to elucidate, understand, and engineer
these pathways so that they can fully harness the wealth of compounds
and biocatalysts that plants have provided.
4. 4
Terpenoid Indole Alkaloids
Are a diverse class of natural products, comprising over 2000 members.
Terpene indole alkaloids are used as anticancer, anti-malarial and anti-arrhythmic agents
All terpene indole alkaloids are derived from tryptophan.
Akuammine tabersonine
Catharanthus roseus
5. 5
Iridoids
Secondary metabolites found in a wide variety of plants and in some animals.
• Intermediates in the biosynthesis of alkaloids
• Consist of a cyclopentane ring fused to a six-membered oxygen heterocycle.
• Found in plants as glycosides.
• Cardiovascular, antihepatotoxic, choleretic, hypoglycemic activities
Iridomyrmex iridomyrmecin
6. Metabolic Pathway Elucidation in 6
Medicinal Plants - Terpene Indole
Alkaloids
• Use bioinformatics to identify potential
genes which code for useful proteins.
• Which genes expressed at known site of
action?
• Test these genes with in vitro assays using
transfection, RNAi and VIGS.
• Goal:
– Improve yield of valuable product/transfect gene
into bacteria/yeast for easier production.
– Adapt new enzymes to expand the library of useful
compounds
7. 7
What is Bioinformatics?
• A powerful tool for
proteomics –
– To use known sequence,
structure, domain or featural
information to predict
features, function or structure
of similar sequence proteins.
8. Identification of a Novel 8
N-Methyltransferase in Vinblastine
Production in Catharanthus roseus
• Knew the stepwise metabolites,
but need to identify an
N-methyltransferase:
• Knew protein was located in
thylakoid membrane.
– Searched the C. roseus genome
for potential methyltransferases
– Select for possible plastidial γ-
tocopherol methyltransferases.
– Select for proteins expressed in
leaves.
S.E. O’Connor et. al. “Homolog of Tocopherol C Methyltransferases Catalyzes N Methylation in Anticancer Alkaloid Biosynthesis”, PNAS, 2010, 107, 44
9. Cytochrome P450 Hydroxylase in 9
Catharanthus roseus:
19-O-acetylhörhammericine production
• Use bioinformatics to identify
proteins likely to interact with
Cytochrome P450
– Discover new proteins involved
in alkaloid biosynthesis.
– Found a unique P450-
dependent enzyme which
stereoselectively hydroxylates
certain alkaloids at a critical
branch point.
S. O’Connor et. al. “Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Caranthus roseus”, J. Bio. Chem, 2011, 286, 19
10. Iridoids – An Alternative Route
to Novel Cyclic Terpenes
O’Connor’s Aims: Uses of Iridoids:
• Identify the • Agricultural Control
reductase protein • Anti-cancer compounds
“Iridoid Synthase”
• Anti-inflammatories
• Explore the effects
of different • Anti-bacterial
reductases on • Anti-fungal Pea aphids,
Acyrthosiphon pisum.
10-oxogeranial
A pest of legume
crops, where
traditional insecticides
kill predators and
parasites.
Geranyl Pyrophosphate 10-Oxogeranial
11. 11
Reductive Cyclisation –
Iridoid Synthase Identification
• Enzyme existence known but not
identified
– Bioinformatics to identify candidates.
– Transfection to E. coli for assays.
– Confirmation and proposed mech.
– Fungal reductase used to produce novel
molecule Inverse electron
demand
Diels–Alder
‡
Michael addition
S.E. O’Connor et. al. “An alternative route to cyclic terpenes by reductive cyclization in iridoid biosynthesis”, Nature, 2012, 138, 494
12. 12
The O’Connor Group:
Jacks of Many Trades
• Explore various complex secondary metabolites
in several different organisms.
– Identify unknown proteins for a valuable product.
– Transfect useful metabolosomes into more viable
hosts.
– Adapt enzymes in various schemes to make novel
non-natural products.
– Bioinformatics to identify related enzymes.