126 Pages
English

Metabolite and isotopologue profiling in plants [Elektronische Ressource] : studies on the biosynthesis of terpenoids and alkaloids / Elena Ostrozhenkova

-

Gain access to the library to view online
Learn more

Informations

Published by
Published 01 January 2008
Reads 29
Language English
Document size 2 MB



Lehrstuhl für Biochemie der Technischen Universität München


Metabolite and isotopologue profiling in plants.
Studies on the biosynthesis of terpenoids and alkaloids.


Elena Ostrozhenkova


Vollständiger Abdruck der von der Fakultät für Chemie der Technischen
Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften genehmigten Dissertation.

Vorsitzende:
Univ.-Prof. Dr. S. Weinkauf

Prüfer der Dissertation:
1. Priv.-Doz. Dr. W. Eisenreich
2. Univ.-Prof. Dr. M. Sattler

Die Dissertation wurde am 16.06.2008 bei der Technischen Universität
München eingereicht und durch die Fakultät für Chemie am 08.07.2008
angenommen.































To my parents, my friends, and me



When a thing was new, people said “It is not true”.
Later, when the truth became obvious, people said “Anyway, it is not
important”. And when its importance could not bedenied,
people said, “Anyway it is not new”.

William James (1842-1920).
Experiments were carried out at the Department of Biochemistry, Technische
Universität München, Garching from April 2003 to April 2008.
ACKNOWLEDGEMENT

I wish to thank my Doktorvater PD Dr. Wolfgang Eisenreich for suggesting me an
exciting and modern scientific theme; for constant and patient guidance and support; for
numerous useful discussions (on scientific matters and beyond); for opening me the
doors into the world of “big” science, with international conferences and collaborations.
My special thanks to Wolfgang for teaching me NMR spectroscopy.
I am thankful to Prof. Dr. Dr. Adelbert Bacher for the opportunity to spend five
great years at the Lehrstuhl für Organische Chemie und Biochemie and for his helpful
mentorship.
I am very grateful to Prof. Dr. Michael Groll for a friendly atmosphere in the
Lehrstuhl für Biochemie, for his encouraging smiles, and for allowing me to finish my
Ph.D. study.
I am indebted to Dr. Felix Rohdich for offering enzymes for my experiments.
I wish to thank Mr. Fitz Wendling (Computer-Man) for many beautiful pictures,
for his professional assistance with computer and HPLC problems, and for familiarizing
me with ´die hochbayrische Sprache´.
I am thankful to Mr. Richard Feicht, Mrs. Christine Schwarz, Ms. Birgit Keil, Ms.
Katrin Gärtner, Mrs. Astrid König, Dr. Melissa Poynor, and Mrs. Ute Kashoa for taking
care of my flowers while I was away and for creating and maintaining a nice and
friendly atmosphere in the lab. My special thanks to Mrs. Christine Schwarz and Ms.
Birgit Keil for HPLC and NMR analysis.
I am indebted to Dr. Tanja Radykewicz, Dr. Ralf Laupitz, Dr. Lilla-Margl-
Römisch, Dr. Werner Römisch-Margl, Ms. Silke Marsch, Dr. Monika Joshi for their
friendly attitude towards me, and just for being around.
I want to thank Ms. Susan Lauw and Mr. Matthias Lee for helping me to prepare
my thesis and concomitant documents, for their assistance in computer matters, and for
their friendship.
I am grateful to Dr. Victoria Illarionova and Dr. Boris Illarionov for their great
help during my first months in Germany, for their constant friendly support, and for not
allowing me to forget the Russian.
It is a pleasure to thank Dr. Stefan Hecht for introducing me into NMR technique,
for his patience, and for helping me along.
I thank Ms. Eva Eylert and Dr. Claudia Huber for assisting me with the analysis of
my probes by GC-MS spectrometry and for their friendly attitude.
My sincere thanks to Mr. Christoph Graßberger for maintaining a warm and
friendly atmosphere in our lab, for helping me with many things, for amazing
philosophical disputes, and for everyday encouraging ´Grüß Gott´.
Thanks a lot to Dr. Nicholas Schramek for his collaboration.
Many thanks to Hans-Fischer Gesellschaft and Phytochemical Society of Europe
(PSE) for their financial support.
It is my please to thank Dr. Leo Jirovetz (University of Vienna, Austria) for a nice
friendship, permanent support, and for constant desire to help.
My very special thanks to ´Frau Professor´ Rosa Cusido, Prof. Javier Palazón, and
Prof. Mercè Bonfill (University of Barcelona, Spain) for the great time in Barcelona and
for our fruitful collaboration, which (I strongly believe) will continue.
Dear Rosa! I am so happy that our virtual acquaintance in 2002 has become real
and personal in 2007!
I am greateful to Prof. Avi Golan (Ben Gurion University of Negev, Israel) for an
interesting and fruitful year of joint work. I am looking forward for our future
collaboration.
I want to say a lot of thanks to Prof. Dr. Maike Petersen (Philipps-Universität
Marburg, Germany). Dear Maike! I am so happy that I can call you my Friend!
I am thankful to my dear friend in metabolomics, Prof. Dr. Rob Verpoorte. Dear
Rob! Thank you so much for my (our) talk in Cambridge in 2007. As you said me there,
“If you are afraid now, you will be afraid forever!” This saying helps me ever since,
thank you for believing in me. I deeply thank you for wonderful years of our contacts,
for our conversations and meetings, and for our victories!
I’m very grateful to my friends in Munich Dr. Gagik Gurzadyan (Gagarin), Dr.
Anatoly Zharikov (Tolik), Dr. Andrey Susha (Andryuha), Dr. Olga Manoilova (Olik).
My special thanks to Dr. Maxim Gelin (Maximo). Lots of thanks to all my friends in
Russia and elsewhere.
My most sincere thanks to my parents and my family for your love, support,
patience, and believing in me.



Publications
Illarionova, V., Kaiser, J., Ostrozhenkova, E., Bacher, A., Fischer, M., Eisenreich, W.,
Rohdich, F., 2006. Nonmevalonate terpene biosynthesis enzymes as antiinfective drug
targets: substrate synthesis and high-throughput methods. J. Org. Chem. 71, 8824-8834.

Kaiser, J., Yassin, M., Prakash, S., Safi, N., Agami, M., Lauw, S., Ostrozhenkova, E.,
Bacher, A., Rohdich, F., Eisenreich, W., Safi, J., Golan-Goldhirsh, A., 2007. Anti-
malerial drug targets: screening for inhibitors of 2C-methyl-D-erythritol 4-phosphate
synthase (IspC protein) in Mediterranean plants. Phytomedicine 14, 242-249.

Ostrozhenkova, E., Eylert, E., Schramek, N., Golan-Goldhirsh, A., Bacher, A.,
Eisenreich, W., 2007. Biosynthesis of the chromogen hermidin from Mercurialis annua
L. Phytochemistry 68, 2816-2824.

Posters and lectures
thThe 9 International Congress “Phytopharm 2005 and PSE”, 2005. Saint-Petersburg,
Russia. (Poster presentation).

A Young Scientists Symposium “Future Trends in Phytochemistry”, 2006. Olomouc,
Czech Republic. (Poster presentation).

Congress “50 Years of the Phytochemical Society of Europe, 2007. Cambridge, UK.
(Lecture and poster presentation).

Invited lecture “Biosynthetic flux in plants containing terpenes and alkaloids. Studies
13 13with CO and [U- C ]glucose”, 2007. Barcelona, Spain. 2 6

A Young Scientists Symposium “Future Trends in Phytochemistry Compounds –
Enzymes – Genes”, 2008. Bad Herrenalb, Germany. (Poster presentation).

Partnering – Workshop “Plant – KBBE”, 2008. Berlin, Germany. (Participation).

GA Symposium “Plant system biology and medicinal plants”, 2008. Leiden,
Netherlands. (Poster presentation).
Table of contens

11. Introduction ………………………………………………………….
1.1. Metabolomics and metabolite profiling …………………………... 1
1.2. Biosynthetic pathways and fluxes …………………………………. 3
1.3. The methods to determine the metabolic pathways and fluxes …. 12
1.4. Objectives …………………………………………………………… 16
172. Materials and Methods ……………………………………………
2.1. Instruments …………………………………………………………. 17
182.2. Materials ……………………………………………………………
2.2.1. Chemicals …………………………………………………………. 18
2.2.2. Enzymes …………………………………………………………… 19
2.2.3. Media for separation ………………………………………………. 20
2.2.4. Spray reagents …………………………………………………….. 21
2.2.5.1. Medium for Nicotiana tabacum………………………………... 21
2.2.5.2. Medium for Allium schoenoprasum……………………………. 22
2.2.6. Plants ………………………………………………………………. 22
2.2.6.1. Allium schoenoprasum ……………………………………….. 22
2.2.6.2. Hypericum perforatum
2.2.6.3. Mercurialis annua L. …………………………………………. 23
2.2.6.4. Nicotiana tabacum, var. havanna piperita …………………… 23
2.2.6.5. Salvia divinorum ……………………………………………… 23
232.3. Methods …………………………………………………………….
2.3.1. Experiments with Allium schoenoprasum ………………………… 23
2.3.2. Metabolite profiling in Allium schoenoprasum …………………… 24
2.3.3. NMR spectroscopy ………………………………………………… 24
2.3.4. Experiments with Hypericum perforatum …………………………. 24
2.3.5. Isolaton of hyperforin ……………………………………………… 25
2.3.6. NMR Spectroscopy ………………………………………………... 25
2.3.7. Isotopologue abundance by NMR spectroscopy …………………... 25
2.3.8. Protein hydrolysis and amino acid derivatization …………………. 27
2.3.9. Mass spectrometry ………………………………………………… 27
2.3.10. Isotopologue abundances by GC-MS ……………………………. 27
2.3.11. Experiments with Mercurialis annua L. …………………………. 29
2.3.11.1. Metabolite profiling in M. annua …………………………….. 29
13 2.3.11.2. Experiments with [U- C ]glucose …………………………… 296
13 2.3.11.3. Experiments with CO ……………………………………... 292
13 2.3.11.4. Experiments with [1- C301
13 15 2.3.11.5. Experiments with [ C , N ]aspartate ……………………… 304 1
2.3.11.6. Isolation of hermidin ………………………………………… 30
2.3.11.7. NMR spectroscopy …………………………………………… 30
2.3.12. Experiments with Nicotiana tabacum ……………………………. 30
2.3.13. Isolation of nicotine ……………………………………………. 30
2.3.14. NMR spectroscopy ………………………………………………. 31
2.3.15. Isolation of glucose ………………………………………………. 31
2.3.16. Isolation of glucose from cellulose ………………………………. 32
2.3.17. NMR spectroscopy ……………………………………………….. 32
2.3.18. Experiments with Salvia divinorum ……………………………… 32
13 2.3.18.1. Experiments with [U- C ]glucose …………………………… 326
13 2.3.18.2. Experiments with CO…………………………………….. 322
2.3.18.3. Isolation of salvinorin A ……………………………………... 33
2.3.18.4. Purification of salvinorin A with column chromatography…... 33
2.3.18.5. Purification of salvinorin A with HPLC …………………….. 33
2.3.18.6. Protein hydrolysis and amino acid derivatization …………… 33
2.3.19. Isolation of amino acids ………………………………………….. 33
2.3.20. Identification of amino acids …………………………………….. 34
2.3.21. Enzymatic syntheses ……………………………………………... 35
13 2.3.21.1. Preparation of [3,4,5- C ]1-Deoxy-D-xylulose 5-phosphate ... 353
13 2.3.21.2. Preparation of [1,3,4- C ]2C-Methyl-D-erythritol 4- 353
phosphate …………………………………………………………………..
13 2.3.21.3. Preparation of [1,3,4- C ]4-Diphosphocytidyl-2C-methyl-D- 363
erythritol …………………………………………………………………..
13 2.3.21.4. Preparation of [1,3,4- CC-methyl-D- 363
erythritol 2-phosphate ………………………………………………………
373. Results and discussion …………………………………………….
3.1. Biological objects …………………………………………………… 37
3.1.1. Allium schoenoprasum L. ……………………………………….. 37
3.1.2. Hypericum perforatum L. ……………………………………….. 38
3.1.3. Mercurialis annua L. ……………………………………………. 39
3.1.4. Nicotiana tabacum ……………………………………………… 40
3.1.5. Salvia divinorum ……………………………………………….. 41
3.2. Metabolite profiling ………………………………………………… 42
3.2.1. Metabolite profiling in Allium schoenoprasum …………………. 42
3.2.2. Metabolite profiling in Mercurialis annua ……………………… 47
3.3. Preparation and properties of isotope-labelled 4-diphosphocytidyl- 52
2C-methyl-D-erythritol 2-phosphate ………………………………………
3.4. Biosynthesis of hyperforin in Hypericum perforatum: experiment 59
13with CO ………………………………………………………………… 2
13 3.4. Use of CO as metabolic tracer. Biosynthesis of salvinorin A ……. 662
3.5. Isotopolog perturbation-relaxation experiments with growing 72
Nicotiana tabacum …………………………………………………………
3.5. Biosynthesis of the chromogen hermidin from Mercurialis annua …. 74
13 3.5.1. Experiments with [U- C ]glucose ……………………………… 746
13 3.5.2. Experiments with CO…………………………………………. 802
13 3.5.3. Experiments with [1- C ]glucose ………………………………. 831
13 15 3.5.4. Experiments with [ C , N ]aspartate …………………………. 884 1
904. Summary
94References