Staff Members of the Institute of Biochemistry, TU Graz http://www ...

Staff Members of the Institute of Biochemistry, TU Graz 

http://www.biochemistry.tugraz.at/ 

Professors 

Peter Macheroux (Full Professor & Head of the Institute) 

(peter.macheroux@tugraz.at; Tel.: +43-(0)316-873-6450) 

Günther Daum (Associate Professor) 

(guenther.daum@tugraz.at; Tel.: +43-(0)316-873-6462) 

Albin Hermetter (Associate Professor) 

(albin.hermetter@tugraz.at; Tel.: +43-(0)316-873-6457) 

Assistants 

Dr. Karin Athenstaedt (until 9.12.2008) 

(karin.athenstaedt@tugraz.at, Tel.:+43-(0)316-873-6460) 

DI Alexandra Binter (since 1.09.2008) 

(alexandra.binter@tugraz.at, Tel.: +43-(0)316-873-6453 

DI Silvia Wallner (since 1.10.2008) 

(silvia.wallner@tugraz.at, Tel.: +43-(0)316-873-6955) 

Office 

Annemarie Portschy 

portschy@tugraz.at; Tel.: +43-(0)316-873-6451; Fax: +43-(0)316-873-6952 

Technical Staff 

Claudia Hrastnik; claudia.hrastnik@tugraz.at; Tel.: +43-(0)316-873-6460 

Martin Puhl; martin.puhl@tugraz.at; Tel.: 43-(0)316-873-6453 

Steve Stipsits; steve.stipsits@tugraz.at; Tel.: 43-(0)316-873-6464 

Rosemarie Trenker-El-Toukhy; r.trenker-el-toukhy@tugraz.at; Tel.: +43-(0)316-873-6464 

Elfriede Zenzmaier; elfriede.zenzmaier@tugraz.at; Tel.: +43-(0)316-873-6467 

Leo Hofer (Workshop); leo.hofer@tugraz.at; Tel.: +43-(0)316-873-8431 or 8433 

1

Brief History of the Institute of Biochemistry 

The Institute of Biochemistry and Food Chemistry was born out of the division of the Institute 

of Biochemical Technology, Food Chemistry and Microchemistry of the former School of 

Technology Graz. Together with all the other Chemistry Institutes, it was located in the old 

Chemistry Building on Baron MANDELL's ground, corner Technikerstrasse-Mandellstrasse. 

1929 The Institute of Technical Biochemistry and Microbiology moved to the building of 

the Fürstlich-Dietrichstein-Stiftung, Schlögelgasse 9, in which all the Bio-Sciences 

were then concentrated. 

1945 Georg GORBACH - initially in the rank of a docent and soon thereafter as a.o. 

Professor - took over to lead the Institute. The Institute was renamed Institute of 

Biochemical Technology and Food Chemistry. 

1948 G. GORBACH was nominated Full Professor and Head of the Institute. In succession 

of the famous Graz School of Microchemistry founded by PREGL and EMICH, Prof. 

GORBACH was one of the most prominent and active leaders in the fields of 

microchemistry, microbiology and nutritional sciences. After World War II, questions 

of water quality and waste water disposal became urgent; hence, the group of the 

future Prof. K. STUNDL, which at that time was part of the Institute, was gaining 

importance. In addition, a division to fight dry-rot supervised by Dr. KUNZE and after 

his demise by H. SALOMON, was also affiliated with the Institute. 

1955 In honour of the founder of Microchemistry and former Professor of the Technical 

University Graz, the extended laboratory was called EMICH-Laboratories. At the same 

time, the Institute was renamed Institute of Biochemical Technology, Food Chemistry 

and Microchemistry. 

Lectures and laboratory courses were held in Biochemistry, Biochemical Technology, Food 

Chemistry and Food Technology, Technical Microscopy and Microchemistry. In addition, the 

Institute covered Technical Microbiology together with Biological and Bacteriological 

Analysis - with the exception of Pathogenic Microorganisms - and a lecture in Organic Raw 

Materials Sciences. 

1970 After the decease of Prof. GORBACH, Prof. GRUBITSCH was appointed Head of the 

Institute. Towards the end of the sixties, the division for water and waste water 

disposal headed by Prof. STUNDL was drawn out of the Institute and established as an 

Institute of its own. Prof. SPITZY was nominated Professor of General Chemistry, 

Micro- and Radiochemistry. This division was also drawn out of the mother Institute 

and at the end of the sixties moved to a new building. 

1973 Division of the Institute for Biochemical Technology, Food Technology and 

Microchemistry took place. At first, Biochemical Technology together with Food 

Technology formed a new Institute now called Institute of Biotechnology and Food 

Chemistry to which the newly nominated Prof. LAFFERTY was appointed Head of the 

Institute. 

2

1973 Dr. F. PALTAUF, docent of the Karl-Franzens-University Graz, was nominated o. 

Professor and Head of the newly established Institute of Biochemistry. The interest of 

Prof. PALTAUF in studying biological membranes and lipids laid the foundation for 

the future direction of research at the Institute. G. DAUM, S. D. KOHLWEIN, and A. 

HERMETTER joined the Institute. All three young scientists were given the chance to 

work as post docs in renowned laboratories in Switzerland and the USA: G. DAUM 

with the groups of G. Schatz (Basel, Switzerland) and R. Schekman (Berkeley, USA), 

A. HERMETTER with J. R. Lakowicz (Baltimore, USA) and S. D. KOHLWEIN with 

S. A. Henry (New York, USA). Consequently, independent research groups 

specialized in cell biology (G. D.), biophysics (A. H.) and molecular biology (S. D. K.) 

evolved at the Institute in Graz, with the group of Prof. F. PALTAUF still focusing on 

the chemistry and biochemistry of lipids. 

Teaching was always a major task of the Institute. Lectures, seminars and laboratory courses 

in basic biochemistry were complemented by special lectures, seminars, and courses held by 

the assistants who became docents in 1985 (G. D.), 1987 (A. H.), and 1992 (S. D. K.). 

Lectures in food chemistry and food technology were held by C. WEBER and H. SALOMON, 

who were staff members of the Institute. Hence the Institute was renamed Institute of 

Biochemistry and Food Chemistry. 

1990 The Institute moved to a new building at Petersgasse 12. Expansion of the size of 

individual research groups and acquisition of new equipment were essential for the 

Institute to participate in novel collaborative efforts at the national and the 

international level including joint projects (Forschungsschwerpunkte, Spezial- 

Forschungsbereiche) and EU-projects. Thus, the Institute of Biochemistry, together 

with partner institutes from the Karl-Franzens-University was the driving force to 

establish Graz as a centre of competence in lipid research. 

1993 W. PFANNHAUSER was appointed as Professor of Food Chemistry. Through his 

own enthusiasm and engagement and that of his collaborators, this new section of the 

Institute rapidly developed and offered students additional opportunities to receive a 

timely education. 

2000 The two sections, Biochemistry and Food Chemistry, being independent of each other 

with respect to personnel, teaching, and research, were separated. The new Institute of 

Biochemistry (Head Prof. PALTAUF) and the new Institute of Food Chemistry and 

Food Technology (Head Prof. PFANNHAUSER) evolved. 

2001 F. PALTAUF, who had been Full Professor of Biochemistry and Head of the 

Department for 28 years, retired in September 2001. G. DAUM was elected Head of 

the Department. S. D. KOHLWEIN was appointed Full Professor of Biochemistry at 

the Karl-Franzens University Graz and left the Institute of Biochemistry of the TU 

Graz. 

2003 P. MACHEROUX was appointed Full Professor of Biochemistry in September 2003 

and Head of the Institute of Biochemistry in January 2004. His research interests 

revolve around topics in the area of protein biochemistry and enzymology which shall 

strengthen the already existing activities at the Graz University of Technology in this 

field (SFB Biocatalysis & Kplus). 

3

Highlights of 2008 

After the successful hearing in January, the FWF-funded PhD program “Molecular 

Enzymology” was approved for a second period (2008-2011) increasing the budget to 3.7 

Million Euro for the 20 laboratories at the participating universities (Karl-Franzens University 

and Graz University of Technology). In the meantime, the number of students in the program 

has risen to 48 with nine PhD students being trained at our institute. 

In June, the group of Peter Macheroux participated at the 16 th International Symposium on 

Flavins and Flavoproteins in Jaca, Spain. Six posters were presented at the conference with 

Sonja Sollner’s poster winning one of six Vincent Massey Poster Awards! The passed year 

has also seen some important publications: In collaboration with Karl Gruber’s group and our 

partner laboratory at the Ruder Boskovic Institute in Zagreb, we have published the first 

crystal structure of dipeptidylpeptidase III. Pravas Baral, a PhD student in Karl’s group, 

successfully crystallized the protein generated by Sigrid Deller and Nina Jajcanin-Jozic in our 

laboratory. The publication was selected best paper of the week by the editors of the Journal 

of Biological Chemistry. In December, Andreas Winkler’s study on berberine bridge enzyme 

was published in the journal Nature Chemical Biology. As before, this work was 

accomplished by a close collaboration in the PhD program “Molecular Enzymology” giving 

Andreas the opportunity to benefit from the expertise in structural biology in Karl’s group 

while carrying out biochemical and biophysical experiments in our institute. 

As in previous years, research in the laboratory of Günther Daum was supported by grants of 

the Austrian Science Fund (FWF). In 2008, a new project devoted to “Phosphatidylserine 

Decarboxylases of the Yeast” was approved. During 2008, a number of new international 

collaborations were established which will be of mutual benefit in the future. Teaching and 

administrative activities of G. Daum included work as chairman of the Doctoral School 

„Molecular Biochemistry and Biotechnology“ at the TU Graz, Board Member of the FWF, 

Associate Editor of FEMS Yeast Research, and Member of the Editorial Board of the JBC. 

Planning of the upcoming Euro Fed Lipid (European Federation for the Science and 

Technology of Lipids) Conference to be held in October 2009 in Graz under the chairmanship 

of G. Daum was another effort during the last year. This conference with 700 expected 

participants will be an important event for the lipid community in Graz. 

Basic research in Albin Hermetter’s group was performed in the framework of two joint 

projects (speaker R. Zechner, IMB, University of Graz) focusing on lipid (patho)physiology. 

His contribution to the GOLD project (GEN-AU program of the BM.W_F) is devoted to the 

functional proteomic analysis of lipases relevant to lipid-associated disorders. Financial 

support of this project has just recently been approved for another period of three years. In the 

SFB Lipotox (FWF project), A. Hermetter studies the toxicity of oxidized phospholipids in 

the cells of the arterial wall. Applied research in lipid enzymology is performed in the Kplus 

center Applied Biocatalysis and will be continued in the planned Austrian Center for 

Industrial Biotechnology (ACIB, speaker A. Glieder, TU Graz) to be established in Graz and 

Vienna in 2010. 

Karin Athenstaedt, completed her “Habilitation” in 2007 and was appointed Docent, 

continued her scientific work on microbial lipid metabolism. She also continued her 

international collaborations with research groups in her field. In October 2008, her grant 

application was approved by the FWF, which will be the starting point for Karin to launch her 

own independent research group in our institute. As in the years before, Karin substantially 

contributed to our teaching activities in biochemistry and molecular biology. 

4

Biochemistry group 

Group leader: Peter Macheroux 

Secretary: Annemarie Portschy 

Senior Research Scientists: Sigrid Deller (until March 31 st ), Andrea Wagner (March-June) 

PhD students: Asma Asghar (until September 30), Alexandra Binter, Heidi Ehammer (until 

July 31), Karlheinz Flicker (until January 31), Martina Neuwirth (until May 31), Sonja 

Sollner, Gernot Rauch (until July 31), Silvia Wallner (since October 1), Andreas Winkler 

Diploma students: Katharina Fallmann, Tanja Knaus, Daniel Koller, Kerstin Motz, Martin 

Poms, Sabrina Riedl, Markus Schober, Silvia Wallner 

Guest student: Sebastian Hofzumahaus (FH Jülich, Germany) 

Technicians: Eva Maria Pointner (karenziert), Martin Puhl (Karenzvertretung), Steve Stipsits 

(Karenzvertretung), Rosemarie Trenker-El-Toukhy 

General description 

The fundamental questions in the study of enzymes, the bio-catalysts of all living organisms, 

revolve around their ability to select a substrate (substrate specificity) and subject this 

substrate to a predetermined chemical reaction (reaction and regio-specificity). In general, 

only a few amino acid residues in the "active site" of an enzyme are involved in this process 

and hence provide the key to the processes taking place during enzyme catalysis. Therefore 

the focus of our research is to achieve a deeper understanding of the functional role of amino 

acids in the active site of enzymes with regard to substrate recognition and stereo- and 

regiospecificity of the chemical transformation. In addition, we are also interested in 

substrate-triggered conformational changes and how enzymes utilize cofactors (flavin, 

nicotinamide) to achieve catalysis. Towards these aims we employ a multidisciplinary 

approach encompassing kinetic, thermodynamic, spectroscopic and structural techniques. In 

addition, we use site-directed mutagenesis to generate mutant enzymes to probe their 

functional role in the before mentioned processes. Furthermore, we collaborate with our 

partners in academia and industry to develop inhibitors for enzymes, which can yield 

important new insights into enzyme mechanisms and can be useful as potential lead 

compounds in the design of new drugs. 

In our laboratory we have established kinetic (stopped-flow and rapid quench analysis of 

enzymatic reactions), thermodynamic (isothermal titration microcalorimetry) and 

spectroscopic (fluorescence, circular dichroism and UV/Vis absorbance) methods. We make 

also frequent use of MALDI-TOF and ESI mass spectrometry, protein purification techniques 

(chromatography and electrophoresis) and modern molecular biology methods to clone and 

express genes of interest to us. A brief description of our current research projects is given 

below. 

Berberine bridge enzyme 

Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a 

pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD 

moiety, which is essential for catalysis. The reaction involves the oxidation of the N-methyl 

group of the substrate (S)-reticuline by the enzyme-bound flavin and concomitant formation of 

a carbon-carbon bond (the “bridge”). The ultimate acceptor of the substrate-derived electrons 

is dioxygen, which reoxidizes the flavin to its resting state: 

5

The BBE-catalysed oxidative carbon-carbon bond formation is a new example of the 

versatility of the flavin cofactor in biochemical reactions. Our goal is to understand the 

oxidative cyclization reaction by a biochemical and structural approach. 

Recently, we have developed a new expression system for BBE (using cDNA from 

Eschscholzia california, gold poppy) in Pichia pastoris, which produces large amounts of the 

protein (ca. 500 mg from a 10-L culture). This work was accomplished in collaboration with 

Prof. Toni Kutchan (Donald Danforth Plant Science Center, U. S. A.) and Prof. Toni Glieder 

(TU Graz). The availability of suitable quantities of BBE enabled us to crystallize the protein 

and to solve the structure in collaboration with Prof. Karl Gruber at the Karl-Franzens 

University Graz (see below). 

Based on the three-dimensional structure of BBE, we have performed a site-directed 

mutagenesis program to investigate the role of amino acids present in the active site of the 

enzyme. In conjunction with other experiments, this has led to the formulation of a new 

reaction mechanism for the enzyme (thesis project of Andreas Winkler; diploma project of 

Sabrina Riedl and Kerstin Motz). 

6

Chorismate synthase 

Chorismate synthase is the seventh enzyme of the shikimate pathway, which leads to aromatic 

amino acids and other aromatic metabolites. Chorismate synthase catalyses the conversion of 

5-enolpyruvylshikimate-3-phosphate to chorismate. The catalytic reaction requires reduced 

flavin for activity. This finding is very intriguing since flavins are essential cofactors in redox 

reactions and the elimination catalysed by chorismate synthase does not (formally) involve a 

reduction or oxidation of the substrate. 

Understanding the mechanism of chorismate synthase is of great importance because, as an 

enzyme of the shikimate pathway, it is only present in bacteria, fungi and plants which makes 

the enzyme an attractive target for the development of new antibiotics, fungicides and 

herbicides. More recently, the shikimate pathway was also discovered in apicomplexan 

parasites (Toxoplasma gondii, Plasmodium falciparum) emphasizing the important role of this 

pathway as a potential drug target. Therefore a mechanistic and structural investigation of 

chorismate synthase may provide valuable information for the development of compounds 

with antimicrobial, fungicidal, herbicidal or antimalarial activity. The mechanistic studies are 

currently concentrating on a site-directed mutagenesis program with the aim to understand the 

role of invariant amino acid residues in the active site of the enzyme. With this approach, we 

expect to gain further insight into the functioning of the enzyme active site and the chemical 

reaction mechanism (thesis project of Gernot Rauch). 

Chorismate synthases can be classified according to the mode of reduction of the required 

cofactor: monofunctional chorismate synthase requires an external source of reduced FMN 

whereas bifunctional enzymes possess an intrinsic NADPH:FMN oxidoreductase activity. The 

latter type of chorismate synthase has - so far - only been found in unicellular fungi (e.g. 

Saccharomyces cerevisiae and Neurospora crassa) and green algae (e.g. Euglena gracilis). 

Our current interest with regard to mono/bi-functionality of chorismate synthases focuses on 

the question whether monofunctional chorismate synthases complement chorismate synthase 

deficient yeast (∆aro2). In order to tackle this issue, we have developed a system to evaluate 

complementation by monofunctional bacterial and plant chorismate synthases in a ∆aro2 yeast 

strain. Our results indicate that bacterial and plant chorismate synthases are unable to confer 

prototrophy to the yeast knock-out despite expression of the proteins (see figure below, central 

panel; Ec, Escherichia coli; An, Anabaena; Le, Lycopersicon esculentum; Bs, Bacillus 

subtilis; Sc, Saccharomyces cerevisiae; Nc, Neurospora crassa; Tt, Tetrahymena 

thermophila). This result suggests that the ability of chorismate synthase to utilize NADPH is 

essential for a fully functional shikimate pathway. This hypothesis receives further support by 

additional experiments where co-complementation with a bacterial NADPH:FMN 

oxidoreductase (YcnD) led to restoration of prototrophy even in the case of monofunctional 

chorismate synthases (see figure below, right panel) (thesis project of Heidi Ehammer). 

7

Dipeptidylpeptidase III 

Dipeptidyl-peptidases III (DPPIII; EC 3.4.14.4) are zinc-dependent enzymes with molecular 

masses of ca. 80-85 kDa that specifically cleave the first two amino acids from the N-terminus 

of different length peptides. All known DPPIII sequences contain the unique motif HEXXGH, 

which enabled the recognition of the dipeptidyl-peptidase III family as a distinct evolutionary 

metallopeptidase family (M49). In mammals, DPPIII is associated with important 

physiological functions such as pain regulation and hence the enzyme is a potential drug 

target. Sigrid Deller and Nina Jajcanin-Jozic have successfully expressed, purified and 

characterized the recombinant yeast enzyme and Pravas Baral in Karl Gruber’s laboratory at 

the Karl-Franzens-University has elucidated the crystal structure of the protein. This work 

revealed that yeast DPPIII is the first representative of a novel protein topology: 

We are now focusing on structures with potential substrates or inhibitors bound in the active 

site. Towards that goal, we are generating inactive mutant proteins to enable cocrystallisation 

with peptide substrates. In addition, we have produced recombinant human DPPIII and are in 

the process to determine the crystal structure to provide the basis for the development of 

potentially useful inhibitors of the enzyme (Gustavo Arruda’s thesis project in Karl Gruber’s 

laboratory assisted by Alexandra Binter) 

Nikkomycin biosynthesis 

Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, 

insecticidal and acaricidal properties due to their strong inhibition of chitin synthase. 

Nikkomycins are promising compounds in the cure of the immunosuppressed, such as AIDS 

patients and organ transplant recipients as well as cancer patients undergoing chemotherapy. 

Nikkomycin Z (R 1 = uracil & R 2 = OH, see below) is currently in clinical trial for its antifungal 

activity. Structurally, nikkomycins can be classified as peptidyl nucleosides containing two 

unusual amino acids, i.e. hydroxypyridylhomothreonine (HPHT) and aminohexuronic acid 

with an N-glycosidically linked base: 

8

Although the chemical structures of nikkomycins have been known since the 1970s, only a 

few biosynthetic steps have been investigated in detail. The steps leading to the 

aminohexuronic acid intermediate are unclear. Originally, it was hypothesized that the 

aminohexuronic acid moiety is generated by addition of an enolpyruvyl moiety from 

phosphoenolpyruvate (PEP) to either the uridine or the 4-formyl-4-imidazolin-2-one analog to 

the 5’-position of ribose. This step is then followed by rather speculative modifications to 

yield the aminohexuronic acid precursor. In contrast to this hypothesis, we could recently 

demonstrate that UMP rather than uridine serves as the acceptor for the enolpyruvyl moiety, a 

reaction catalyzed by an enzyme encoded by a gene of the nikkomycin operon termed nikO. 

Furthermore, we could demonstrate that it is attached to the 3’- rather than the 5’-position of 

UMP. These results are very intriguing since none of the nikkomycins synthesized possess an 

enolpyruvyl group in this position of the sugar moiety. Hence, it must be concluded that the 

resulting 3’-enolpyruvyl-UMP is subject to rearrangement reactions where the enolpyruvyl is 

detached from its 3’-position and transferred to the 5’-position of the ensuing aminohexuronic 

acid moiety. Currently, nothing is known about the enzymes involved in these putative 

chemical steps. However, the genes that are co-transcribed with nikO have been reported and 

are designated nikI, nikJ, nikK, nikL, nikM and nikN. In order to investigate the role of the 

encoded proteins in the biosynthesis of the aminohexuronic acid moiety, these genes will be 

cloned, expressed and the proteins purified for biochemical characterization (thesis project of 

Alexandra Binter; diploma project of Sebastian Hofzumahaus). 

Proteins in a new vitamin B 6 biosynthetic pathway 

Vitamin B 6 (pyridoxine) is an essential nutritional compound required by animals and 

humans, which lack the biosynthetic pathway for its production. Only bacteria, fungi and 

plants possess the capability to synthesise vitamin B 6 from basic sugar components. The 

chemistry and biochemistry of the vitamin B 6 biosynthesis has been studied in depth in the 

model bacterium Escherichia coli and it has been thought that this pathway is shared by all 

other organisms in which vitamin B 6 biosynthesis occurs. In recent years, however, it emerged 

that the biosynthetic pathway found in E. coli is limited to a small group of eubacteria (chiefly 

the γ-subdivision) and the majority of organisms have developed an entirely different route to 

generate vitamin B 6 . 

In our studies, we focus on two new proteins that are associated with the biosynthesis of 

vitamin B 6 in the prokaryote Bacillus subtilis: Pdx1 and Pdx2. The major goal of our studies 

is to unravel their interaction which results in the generation of pyridoxal phosphate. In order 

9

to achieve this, both proteins have been recombinantly produced and purified in quantities that 

afford the characterisation of binding by isothermal titration calorimetry. 

Moreover, we have cloned and expressed the Pdx1 homolog Snz1 from Saccharomyces 

cerevisiae. The purified protein was crystallized and the three-dimensional structure solved by 

x-ray crystallography in collaboration with Dr. Ivo Tews, Biochemiezentrum Heidelberg. 

Interestingly, it was found that Snz1 exists as a hexamer rather than a dodecamer (see scheme 

below) and we are currently identifying the structural features that control oligomerization in 

the Pdx1 domain (thesis project of Martina Neuwirth and diploma project of Silvia Wallner). 

The absence of vitamin B 6 biosynthesis in humans clearly suggests that the proteins involved 

are interesting new drug targets for the development of antibiotics, fungicides and herbicides. 

The recent discovery of vitamin B 6 biosynthesis in the causative agent of malaria, Plasmodium 

falciparum, prompted us to engage in an international collaboration in order to characterise 

the chemistry and enzymology of vitamin B 6 biosynthesis in this highly pathogenic organism. 

Our studies will provide a detailed understanding of the role(s) of Pdx1 and Pdx2 in P. 

falciparum and will enable us to initiate a drug design/screening programme which will 

receive additional support from the elucidation of the three-dimensional structures of the 

proteins and their active complex (thesis project of Karlheinz Flicker). 

NAD(P)H:FMN quinone reductases 

Following our mechanistic and structural studies on YcnD, a nitroreductase from Bacillus 

subtilis, we have cloned another NADPH-dependent flavin containing oxidoreductase from 

this organism (termed YhdA). We have achieved high level expression in E. coli BL 21 host 

cells and purified it to homogeneity. Steady-state kinetic studies have shown that YhdA 

reduces FMN and nitro-organic compounds at the expense of NADPH as the preferred 

electron donor. In parallel to the bacterial enzyme, we have also started to investigate the 

properties of a homologous enzyme from Saccharomyces cerevisiae (termed LOT6p). Despite 

the availability of a three-dimensional x-ray structure for YhdA (1NNI) and LOT6p (1T0I), 

the physiological role of the enzyme was unclear. Our recent studies have now demonstrated 

that both enzymes rapidly reduce quinones at the expense of a reduced nicotinamide cofactor. 

In order to further characterize the cellular role of LOT6p, we have carried out pull-down 

10

assays and identified the 20S core particle of the yeast proteasome as a protein interaction 

partner. Further studies revealed that this complex recruits Yap4p, a member of the b-Zip 

transcription factor family, but only when the flavin-cofactor of Lot6p is in its reduced state. 

Oxidation of the flavin leads to dissociation of the transcription factor and relocalization to 

the nucleus. A similar system is known from mammalian cells, where a homologous quinone 

reductase (NQO1) binds to the 20S proteasome and recruits important tumor suppressor 

proteins such as p53 and p73α. Hence, the discovery of a homologous protein interaction in 

yeast provides an interesting model system to investigate the molecular basis for protein 

complex formation and regulation of proteasomal degradation of transcription factors (thesis 

project of Sonja Sollner and diploma project of Markus Schober). 

Diploma theses completed 

Katharina Fallmann: Enzymes of Nikkomycin Biosynthesis: NikJ, NikL, and NikO 

Nikkomycins are peptidyl nucleoside antibiotics that competitively inhibit chitin synthase and 

thus exhibit antifungal and insecticidal properties. The chemical structure of nikkomyins 

consists of the unusual amino acids hydroxypryridylhomothreonine (HPHT) and an 

aminohexuronic acid with, in the case of nikkomycin X, N-glycosidically linked 4-formyl-4- 

imidazolin-2-one (imidazolone base) or, in the case of nikkomycin Z, N-glycosidically linked 

uracil. Nikkomycin biosynthesis in Streptomyces tendae requires a set of proteins which are 

grouped in few operons at the genetic level. Some Nik proteins have already been studied in 

detail, but the complete pathway of nikkomycin biosynthesis is not yet known. In this work, 

the proteins NikJ, NikO, and the heterologous expression of the putative protein product of 

nikL were studied. The nikJ gene was heterologously expressed with a C-terminal polyhistidine 

tag in E. coli. Suitable expression conditions and a purification protocol for the 

protein NikJ were developed at laboratory scale. The expression requires iron 

supplementation of the growth medium in order to facilitate the formation of the iron-sulfur 

clusters during over-expression of NikJ. The optimised purification protocol employs affinity 

chromatography at a pH value of 8.5. The identity of the purified NikJ protein was confirmed 

by MALDI-TOF MS. Analytical gel filtration chromatography of NikJ showed a size between 

dimer and trimer in vitro. The results from UV-Vis spectroscopy indicated the presence of 

coenzymes or prosthetic groups in NikJ. EPR spectroscopy revealed NikJ to be an iron-sulfur 

protein in accordance with the in silico sequence analysis results. For NikO, analytical gel 

filtration chromatography experiments confirmed earlier results on its quaternary structure 

showing a complex of the size between two and three monomers in vitro. Additionally, NikO 

was purified for protein crystallization. The attempts to express the respective protein from 

the nikL gene heterologously in E. coli did not result in any detectable protein amount. 

Possible reasons for the lack of expressed protein were analyzed, ruling out cloning errors by 

sequencing and restriction analysis, as well as expression failure induced by the different 

frequency of codons in the source organism S. tendae and the expression host E. coli, or an 

unfavourable mRNA secondary structure of the transcript, by means of in silico analysis. 

Tanja Knaus: Characterization of the NAD(P)H:FMN Oxidoreductase YcnD from Bacillus 

subtilis 

The results of the presented diploma thesis clearly indicate that the NAD(P)H:FMN 

oxidoreductase YcnD from the gram-positive bacterium Bacillus subtilis, which has 

previously been shown to exhibit azoreductase activity, also has a significant quinone 

reductase activity. Using steady-state kinetic and rapid reaction measurements, reduction of 

11

several quinone substrates was investigated, showing K M values in the low micro molar range. 

For reactions with quinones the reductive half reaction of the redox process was found to be 

the rate-determining step. In comparison to other quinone reductases, YcnD does not have the 

typical flavodoxin-fold, however reduction occurs by a two-electron reduction process, thus 

not stabilizing large amounts of radical semiquinone intermediates. Like many other FMN 

dependent quinone reductases, YcnD is inhibited by dicoumarol. In addition to this, the 

enzyme under investigation shows a sulfite adduct formation with sodium sulfite, being 

typical rather for oxidases than quinone reductases. Apart from these findings necessary for 

enzyme characterization, no clear evidence was found that YcnD is a part of the trimeric 

complex with two enzymes of the shikimate pathway, chorismate synthase and 

dehydroquinate synthase, respectively, by performing various pull-down assays and 

isothermal titration calorimetry measurements. 

Daniel Koller: Die Rolle von NikU und NikV in der Nikkomycinbiosynthese von 

Streptomyces tendae 

Nikkomycins are nucleoside-peptide antibiotics, which have antifungal, insecticidal, and 

acaridal activity, by competitive inhibition of several chitin synthases. These enzymes are 

involved in cell wall synthesis and are inhibited by nikkomycins because of their similarity to 

UDP-N-acetylglucosamine, the natural substrate of these enzymes. Approximately 20 

biologically active nikkomycin structures were isolated from the culture filtrate of 

Streptomyces tendae. The genes involved in biosynthesis of nikkomycins in S. tendae were 

identified and based on the putative activity of the encoded enzymes a hypothetical pathway 

was postulated. The purpose of this diploma thesis was the heterologous expression of two 

proteins of this pathway, NikU and NikV, in their native form in order to obtain sufficient 

material for protein crystallisation and subsequent x-ray analysis. NikU and NikV are subunits 

of a 22-heterotetramer, coenzyme B12-dependent glutamate mutase. Toward that goal, 

modified E. coli BL21 (DE3), E. coli Rosetta and strains containing rare t-RNAs were tested 

for protein expression. However, it appears that the difference in codon usage was 

unfavourable for high-level expression of Streptomyces genes in E. coli. Another problem 

encountered during my study was the formation of inclusion bodies. To avoid this problem, I 

have studied the effect of temperature and coexpression on the solubility of heterologously 

expressed NikU and NikV. At 37 °C NikU is expressed partially native unlike NikV. At 20 °C 

both proteins are generated in a soluble form. In addition, it was found that coexpression did 

not result in a better yield of NikU or NikV at any of the temperatures investigated. In 

summary, the expression levels reached in bacterial host cells used in this diploma thesis were 

not satisfactory and sufficient protein material for crystallization could therefore not be 

obtained. 

Martin Poms: Backbone cyclisation of the conotoxin ρ-TA 

Conotoxins, small, disulfide-rich peptides isolated from the venom of cone snails, have 

created much interest as possible drug leads in the last decades due to their high potency and 

great specificity. Their ability to selectively inhibit ion channels could be valuable for a wide 

range of therapeutical applications, while minimizing possible side effects. The main problem 

of peptides as therapeutics presents itself in the susceptibility to enzymatic degradation. One 

solution to this problem could be the introduction of a cyclic backbone to the peptide to 

increase the stability while maintaining the activity. In this study, two cyclic analogues of the 

conotoxin ρTIA were synthesised and compared to the native peptide in accordance to their 

NMR structure, serum stability and activity towards the 1-adrenergic receptor. It shows that 

12

the cyclic analogues maintain their stability as well as a significant level of activity. Even 

though backbone cyclization has been applied to other conotoxins before, this means that the 

technique of backbone cyclization can be applied to an even wider range of peptides and 

therefore serve as a useful tool in therapeutics development to increase drug stability. 

Sabrina Riedl: Biochemical characterization of BBE mutants 

The berberine bridge enzyme (BBE) is an important enzyme of the alkaloid biosynthesis in 

plants e.g. the California poppy (Eschscholzia californica), the Opium poppy (Papaver 

somniferum) and other members of the Papaveraceae and Berberidaceae. It catalyzes the 

conversion of (S)-reticuline to (S)-scoulerine. Therefore, the transformation of the N-methyl 

group of (S)-reticuline into the berberine bridge carbon (C-8) of (S)-scoulerine is necessary. 

This is achieved by a 2-step mechanism. BBE is a flavoprotein containing a FAD cofactor 

which is bi-covalently attached to the protein via an 8-histidyl and 6-S-cysteinyl linkage. This 

type of bi-covalent linkage was just discovered about two years ago. For this reason only very 

little is known about the influence of the two covalent linkages. To investigate the importance 

of some active site amino acids mutations were introduced into the protein. Some mutations 

concerned the flavinylation of the protein e.g. at the site of covalent attachment or amino acids 

that are suggested to influence the formation of the covalent linkage to the protein. Other 

mutations were made concerning the reaction mechanism e.g. amino acids which might be 

responsible for proton abstraction during the reaction. The mutations were expressed in Pichia 

pastoris host cells and purified using a 2-step purification protocol. They were then analyzed 

regarding enzyme activity and the way of cofactor attachment. Three different mutants were 

analyzed: C166S, H174A and H104A. The H174A mutant is involved in the reaction 

mechanism. Introduction of this mutation led to a decreased enzyme activity of less than 1 % 

compared to the wild-type. The redoxpotential was reduced to 44.0 mV what is a difference of 

88 mV (wild-type: 132 mV). The C166S and the H104A mutation concern the covalent 

attachment to the cofactor. It was shown that serine could not replace cysteine and form the 

covalent linkage which was the point of interest. The H104A mutant also showed a substantial 

decrease of activity (only 6 %) although the redox potential was only slightly affected. This 

led to the conclusion that bi-covalent attachment is necessary for sufficient enzyme activity. 

Markus Schober: The transcription factor Yap4p and its interaction with the quinone 

reductase Lot6p 

In the course of time organisms have evolved complex strategies to counteract oxidative 

stress. These responses are rarely triggered by a single factor but involve a cascade of protein 

interactions, which are the subject of countless studies. Transcriptional regulation and 

proteolytic degradation both play major roles in stress response. The tumor suppressing 

transcription factor p53, whose cellular level is known to be regulated by the ubiquitin-26S 

proteasome-Mdm2 system, was found to be accumulated in times of oxidative stress. It is 

thought that this accumulation is caused by a different ubiquitin- and Mdm2-independent 

pathway, which is regulated by NAD(P)H quinone oxidoreductase 1 (NQO1) that protects p53 

from proteasomal degradation by the 20S proteasome. Recently, a potential interaction 

between the 20S proteasome, the quinone reductase Lot6p and the transcription factor Yap4p, 

a member of the AP1-like yeast activator protein family involved in the oxidative stress 

response was discovered in our research group. This interaction is only occurring when Lot6p 

is present in its reduced form but does not depend on the presence of NADH, which has yet 

not been shown for the p53-20S proteasome-NQO1 pathway. Our in vitro experiments have 

shown that Yap4p is also degraded by the 20S proteasome in an ubiquitin independent 

13

fashion. We hypothesize that Yap4p is protected from proteasomal degradation when it is 

bound to Lot6p or that interaction of Yap4p with Lot6p regulates Yap4p’s localization in the 

cell. 

Silvia Wallner: Thermodynamic interaction studies on the Pdx2/Pdx2 interface of Bacillus 

subtilis. 

Vitamin B6 is a versatile cofactor, which is absolutely required for physical and mental health 

in humans. Today two different pathways for de novo biosynthesis of pyridoxal 5-phosphate 

(PLP) are characterized, which are designated as DXP-dependent and DXP-independent 

pathway. In the predominant route, referred to as DXP-independent pathway, PLP is 

synthesized directly from a pentasaccharide and a trisaccharide in presence of L-glutamine. 

This reaction is catalyzed by a heteromeric glutamine amidotransferase complex consisting of 

12 Pdx1 synthase and 12 Pdx2 glutaminase subunits. In this diploma project various aspects 

of complex formation of the Bacillus subtilis PLP synthase complex have been analyzed. 

Today the crystal structure of the ternary Pdx1:Pdx2H170N:L-glutamine complex and of the 

individual Pdx1 and Pdx2 moieties are available. Moreover, in previous studies isothermal 

titration calorimetry has been applied to determine the thermodynamics of complex formation. 

In order to understand the complex mode of interaction between the B. subtilis Pdx1 and Pdx2 

moieties, the contribution of specific amino acid residues to complex assembly has been 

investigated. Candidates for site-directed mutagenesis have been selected according to 

structural predictions. For this study all chosen amino acid residues concern either Pdx2 

oxyanion hole formation or the correct placing of the Pdx1 N-helix. Thermodynamics of 

complex formation have been evaluated for all generated mutant proteins applying isothermal 

titration calorimetry. Microcalorimetry experiments show that Pdx2 Gln10 actually affects 

oxyanion hole formation and the tightness of the PLP synthase complex. Moreover, 

thermodynamic data approve the importance of Pdx1 Lys18 for stabilization of Pdx1 N. Pdx1 

Lys18 forms polar contacts to Pdx2 Gln10 and Glu15 and hence holds the crucial N-helix in 

place. These new insights in interaction processes on the Pdx1/Pdx2 interface may help to get 

a better understanding of the mode of PLP synthase complex formation. Due to the absence of 

a PLP biosynthetic pathway in humans this pathway could eventually be a potential target for 

new drug development and hence a holistic understanding of the PLP synthase complex is 

required. 

Doctoral theses completed 

Heidemarie Ehammer: Biochemical studies on the enzymology and evolution of the 

shikimate pathway 

The shikimate pathway has been found in plants, bacteria, fungi and some protozoa and is 

essential for the biosynthesis of aromatic compounds. As mammals do not utilise this 

pathway, the enzymes involved can be used as potential targets for the development of new 

antibiotics. Chorismate synthase catalyses the last step of the shikimate pathway that leads to 

the formation of chorismate, which is the branch point metabolite in the synthesis of aromatic 

amino acids and a variety of aromatic compounds. This enzyme has an absolute requirement 

for reduced flavin. There are two classes of chorismate synthases, which are distinguished 

according to the origin of the reduced flavin cofactor. Monofunctional chorismate synthases 

have been identified in plants and bacteria and depend on the level of reduced FMN from the 

cellular environment. However, bifunctional enzymes from fungi and the ciliated protozoan 

Euglena gracilis can generate reduced FMN at the expense of NADPH. The main focus of 

14

this work lies in the classification of hitherto uncharacterised chorismate synthases by 

introducing an in vivo screen, which is based on a chorismate synthase deficient 

Saccharomyces cerevisiae strain. This analysis revealed that bifunctionality is present in 

enzymes of protozoan species such as the parasite Toxoplasma gondii. In contrast, all bacterial 

and plant chorismate synthases tested are monofunctional. In addition, I have demonstrated 

that a monofunctional chorismate synthase confers prototrophy in conjunction with an 

NADPH:FMN oxidoreductase indicating that bifunctionality is required due to the lack of free 

reduced FMN in fungal and possibly protozoan species. Interestingly, I have found that the 

presence of a bifunctional enzyme correlates with the occurrence of a pentafunctional arom 

complex. The conclusion that fungal and protozoan species have a common ancestor is 

consistent with phylogenetic studies of the shikimate pathway. The arom complex comprises 

the enzymes catalysing steps 2 to 6 of the shikimate pathway. Along with the bifunctional 

chorismate synthase, the arom complex is present in fungal and protozoan species whereas in 

bacteria the same reactions are catalysed by five monofunctional enzymes. We have been 

interested in the investigation of the crystal structure of the arom complex from 

Saccharomyces cerevisiae, Aro1p, to get insights into the enzyme mechanism of 

multifunctional complexes. Although a good level of Aro1p expression has been observed in 

E. coli, further investigations will be necessary to isolate higher amounts of pure protein. 

Karlheinz Flicker: Thermodynamic interaction studies on the assembly of heteromeric 

pyridoxal-5’-phosphate synthase from Plasmodium falciparum 

Pyridoxal-5'-phosphate (PLP) is an essential nutrient for animals and humans as it acts as a 

cofactor in many enzymes. The predominant de novo biosynthetic route is catalysed by PLP 

synthase consisting of twelve individual Pdx1/Pdx2 glutamine amidotransferase heterodimers 

in which Pdx1 is the synthase and Pdx2 the glutaminase. In this study, complex formation of 

PLP synthase from the human malaria pathogen Plasmodium falciparum has been 

characterised using isothermal titration calorimetry. The thermodynamic data are integrated in 

a 3D-homology model of plasmodial PLP synthase and are compared in detail with data from 

the previously investigated PLP synthase from B. subtilis. Presence of L-Gln increases the 

affinity of the complex 30-fold and alters the thermodynamic signature of complex formation. 

According to experimental heat capacities the interface in the presence of L-Gln is less 

dominated by hydrophobic interactions than in its presence. Fluorescence studies also verify 

the hydrophobic character of the PfPdx1 protein interface. In addition, the role of the N- 

terminal region of PfPdx1 for complex formation has been verified. A swap mutant in which 

the complete αN-helix of PfPdx1 was exchanged for the corresponding segment from BsPdx1 

shows cross-binding to BsPdx2 and also elicits partial glutaminase activity in BsPdx2. This 

demonstrates that formation of the protein complex interface via αN and catalytic activation 

of the glutaminase are linked processes. 

Martina Neuwirth: Interaction studies on Pdx1 and Pdx2 – two proteins involved in vitamin 

B6 biosynthesis 

Pyridoxal 5-phosphate (PLP), the biologically active form of vitamin B6, is one of the most 

versatile organic cofactors in nature. While animals and humans are not capable to synthesize 

this vitamin, most bacteria, fungi, protozoa, and plants utilize a de novo PLP biosynthesis 

pathway catalyzed by a glutamine amidotransferase. This PLP synthase, composed of a 

synthase- (Pdx1) and a glutaminase-subunit (Pdx2), is not a single heterodimer, but a complex 

of approximately 650 kDa. In this complex twelve Pdx1 molecules form a double-ring shaped 

15

dodecameric core. Each of the twelve Pdx1 subunits independently interacts with one of 

twelve Pdx2 molecules, forming the whole 24meric PLP synthase complex. In this study, 

complex formation of the bacterium Bacillus subtilis was thermodynamically characterized. It 

could be shown that complex formation depends on the presence of the substrate L-glutamine, 

which has a tightening effect on the preformed Pdx1/Pdx2 complex. In other words, L- 

glutamine increases the affinity of the two molecules to each other and alters thermodynamic 

properties of the complex. Furthermore, the contribution of single amino acids located in the 

Pdx1/Pdx2 interface to complex formation, were investigated. Based on the crystal structure 

of the B. subtilis PLP synthase complex, single amino acid residues were exchanged and the 

mutated proteins were analyzed in terms of their thermodynamic and enzymatic properties. 

Correlation of the obtained data with structural data yielded a deeper insight into the reaction 

mechanism related to structural changes upon complex formation. Furthermore, the crystal 

structure of Snz1, the homologues protein to Pdx1 in the fungus Saccharomyces cerevisiae, 

could be solved. Surprisingly, Snz1 did not show the expected dodemcameric form, but 

appeared to be a hexamer assembled in the same manner as the B. subtilis Pdx1 core. It could 

be shown that an additional lysine residue in Snz1, lacking in B. subtilis Pdx1, leads to this 

effect. Moreover, the importance of the C-terminal end, entirely visible in the Snz1 3D 

structure and showing a close vicinity to the enzymes active site, could be demonstrated. Due 

to its absence in humans, PLP biosynthesis, considered to be a potential new drug target, is of 

great interest in the medical field. The new findings described in this study lead to a better 

understanding of the Pdx1/Pdx2 interaction, and thus may contribute to the development of 

new therapeutic strategies. 

Gernot Rauch: Mechanistic studies on chorismate synthase 

Chorismate synthase catalyzes the anti-1,4-elimination of the 3-phosphate and the C(6proR) 

hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, the final 

product of the common shikimate pathway and a precursor for the biosynthesis of aromatic 

compounds. The enzyme has an absolute requirement for reduced FMN, which is thought to 

facilitate cleavage of the C-O bond by transiently donating an electron to the substrate. The 

crystal structure of the enzyme revealed that EPSP is bound near the flavin isoalloxazine ring 

with several invariant amino acid residues in contact with the substrate and/or cofactor. In this 

work I present the results of two mutagenesis studies. In the first study, the invariant aspartate 

367 was replaced through alanine and asparagine. Both single mutant proteins (Asp367Ala 

and Asp367Asn) were comparable to the wild-type enzyme with respect to substrate and 

cofactor binding, indicating that Asp367 is not required for binding of either the flavin or the 

substrate. In sharp contrast to these results, the activity of both single mutant proteins was 

found to be 620 and 310 times lower for the Asp367Ala and Asp367Asn mutant proteins, 

respectively. The data provides the first experimental evidence that the carboxylate group of 

aspartate 367 participates in the deprotonation of N(5) of the reduced flavin cofactor, which in 

turn abstracts the C(6proR) hydrogen yielding chorismate as the product. In the second study 

two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate 

synthase were replaced with alanine, producing two single-mutant proteins (Ser16Ala and 

Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). The residual activity of the 

Ser127Ala and Ser16Ala single-mutant proteins was found to be six- and 70-fold lower, 

respectively, than that of the wild-type protein. No residual activity was detected for the 

Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient 

intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, 

in contrast to the single-mutant proteins. Based on the structure of the enzyme, this study 

provides strong evidence that Ser16 and Ser127 form part of a proton relay system among the 

16

isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for 

the formation of the transient intermediate and for substrate turnover. 

International cooperations 

M. Abramic, Ruder Boskovic Institute Zagreb, Croatia 

B. Kappes, Universitätsklinikum Heidelberg, Germany 

T. Kutchan, Donald Danforth Plant Science Center, U.S.A. 

M. Groll, Technical University Munich, Germany 

B. A. Palfey, University of Michigan, U.S.A. 

I. Tews, Universität Heidelberg, Germany 

Research projects 

FWF P19858: “Enzymes of nikkomycin biosynthesis” 

FWF P17215: “Proteins involved in a novel vitamin B 6 pathway” 

FWF P17471: “Mechanism of chorismate synthase” 

FWF-Doktoratskolleg “Molecular Enzymology” 

Invited lectures 

1) Macheroux, P., Schober, M., Sollner, S. 

The role of quinone reductase as a redox sensor in eukaryotic cells 

16 th International Symposium on Flavins & Flavoproteins, Jaca, Spain, 12.06.2008 

2) Sollner, S., Schober, M., Wagner, A., Prem, A., Deller, S., Groll, M., Palfey, B., 

Macheroux, P. 

Toward an understanding of the cellular role of yeast quinone reductase Lot6p 

Vincent Massey Award Lecture at the 16 th International Symposium on Flavins & 

Flavoproteins, Jaca, Spain, 12.06.2008 

3) Winkler, A., Gruber, K., Macheroux, P. 

Structure function studies on berberine bridge enzyme 

65 th Harden Conference on Enzymes: Nature’s Molecular Machines, University of 

Cumbria, Ambleside, U. K., 17.08.2008 

4) Macheroux, P., Winkler, A., Gruber, K. 

Structure and mechanism of berberine bridge enzyme 

2 nd International Interdisciplinary Conference on Vitamins, Coenzymes & Biofactors, 

Athens, Georgia, U. S. A., 26.10.2008 

Publications 

1) Deller, S., Macheroux, P. and Sollner, S.: 

Flavin-dependent quinone reductases 

Cell. Mol. Life Sci. 2008, 65:141-160. 

17

2) Hall, M., Stueckler, C., Ehammer, H., Pointner, E., Oberdorfer, G., Gruber, K., Hauer, 

B., Stuermer, R., Kroutil, W., Macheroux, P., and Faber, K.: 

Asymmetric bioreduction of C=C bonds using enoate reductases OPR1, OPR3 and 

YqjM: Enzyme-based stereocontrol 

Advanced Synthesis & Catalysis 2008, 350:411-418. 

3) Rauch, G., Ehammer, H., Bornemann, S., and Macheroux, P.: 

Replacement of two invariant serine residues in chorismate synthase provides evidence 

that a proton relay system is essential for catalytic activity 

FEBS Journal 2008, 275:1464-1473. 

4) Ande, S. R., Fussi, H., Knauer, H., Murkovic, M., Ghisla, S., Fröhlich, K.-U., and 

Macheroux, P.: 

Induction of apoptosis in yeast by ophidian L- amino acid oxidase 

Yeast 2008, 25:349-357. 

5) Baral, P. K., Jajcanin-Jozic, N., Deller, S., Macheroux, P., Abramic, M., and Gruber, K.: 

The first structure of dipeptidylpeptidase III (DPP-III) provides insight into the catalytic 

mechanism and the mode of substrate binding 

J. Biol. Chem. 2008, 283:22316-22324. 

6) Huang, C.-H., Winkler, A., Chen, C.-L., Lai, W.-L., Tsai, Y.-C., Macheroux, P., and 

Liaw, S.-H.: 

Functional roles of the 6-S-cysteinyl, 8α-N1-histidyl FAD in glucooligosaccharide 

oxidase from Acremonium strictum 

J. Biol. Chem. 2008, 283:30990-30996. 

7) Voss, C. V., Gruber, C. C., Faber, K., Knaus, T., Macheroux, P., and Kroutil, W.: 

Orchestration of a concurrent oxidation and reduction cycle 

J. Am. Chem. Soc. 2008, 130:13969-13972. 

8) Winkler, A., Lyskowski, A., Riedl, S., Puhl, M., Kutchan, T., Macheroux, P., and 

Gruber, K.: 

A concerted mechanism for berberine bridge enzyme 

Nat. Chem. Biol., 2008, 4:739-741. 

Award 

Sonja Sollner: Vincent Massey Award at the 16 th International Symposium on Flavins and 

Flavoproteins (June 8-13, 2008), Jaca, Spain. 

18

Cell Biology Group 

Group leader: Günther Daum 

Graduate students: Andrea Wagner, Tamara Wriessnegger, Irmgard Schuiki, Tibor Czabany, 

Melanie Connerth, Sona Rajakumari, Karlheinz Grillitsch, Miroslava Spanova, Susanne 

Horvath (since Nov. 2008) 

Diploma student: Susanne Horvath (till Oct. 2008), Sabine Zitzenbacher (since Oct. 2008) 

Technician: Claudia Hrastnik 

Visiting scientists: Meng Lin, Christian Albrecht University, Kiel, Germany 


The existence of functional organelles is the basis for regulated processes within a cell. To 

sequester organelles from their environment, membranes are required which not only protect 

the interior of the organelles but also govern communication with the rest of the cell. 

Therefore, it is very important to understand the biogenesis and maintenance of biological 

membranes. To study this question our laboratory makes use of the yeast as a well established 

experimental system. Our studies are focused on the synthesis of lipids and their assembly into 

yeast organelle membranes. We make use of the advantages of this experimental system to 

combine biochemical, molecular and cell biological methods addressing problems of lipid 

metabolism, lipid depot formation and membrane biogenesis. 

The majority of yeast lipids are synthesized in the endoplasmic reticulum (ER) with some 

significant contributions of mitochondria, the Golgi and the so-called lipid particles. Other 

subcellular fractions are devoid of lipid-synthesizing activities. Spatial separation of lipid 

biosynthetic steps and lack of lipid synthesis in several cellular membranes necessitate 

efficient transfer of lipids from their site of synthesis to their proper destination(s). The 

maintenance of organelle lipid profiles requires strict coordination of biosynthetic and 

translocation activities. Specific aspects studied currently in our laboratory are (i) assembly 

and homeostasis of phosphatidylethanolamine in yeast organelle membranes with emphasis on 

peroxisomes and the plasma membrane, (ii) neutral lipid storage in lipid particles/droplets and 

mobilization of these depots with emphasis on the involvement of lipases and hydrolases, and 

(iii) characterization of organelle membranes from the industrial yeast Pichia pastoris. 

Phosphatidylethanolamine, a key component of yeast organelle membranes 

Work from our laboratory and from other groups had shown that phosphatidylethanolamine 

(PtdEtn), one of the major phospholipids of yeast membranes, is highly important for cellular 

function and cell proliferation. PtdEtn synthesis in the yeast is accomplished by a network of 

reactions including (i) synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum, 

(ii) decarboxylation of PtdSer by mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) 

or (iii) Psd2p in a Golgi/vacuolar compartment, (iv) the CDP-ethanolamine pathway 

(Kennedy pathway) in the endoplasmic reticulum, and (v) the lysophospholipid acylation 

route catalyzed by Ale1p. To obtain more insight into biosynthesis, assembly and homeostasis 

of PtdEtn single and multiple mutants bearing defects in the respective pathways can be used. 

Previous investigations in our laboratory were aimed at the molecular biological 

identification of novel components involved in PtdEtn homeostasis of the yeast Saccharomyces 

cerevisiae. For this purpose, a number of genetic screenings were performed. To 

19

obtain a global view of the role of PtdEtn in the cell and to study the effects of an unbalanced 

PtdEtn level we recently subjected a psd1∆ deletion mutant and the corresponding wild-type 

to DNA microarray analysis and examined genome-wide changes in gene expression caused 

by a PSD1 deletion. Comparison of the gene expression pattern of the psd1∆ mutant with the 

wild-type led to the identification of 55 differentially expressed genes. Grouping of these 

genes into functional categories revealed that PtdEtn formation by Psd1p influenced the 

expression of genes involved in diverse cellular pathways including transport, carbohydrate 

metabolism and stress response. Most of the proteins identified are present in the cytosol 

followed by the nucleus, cellular membranes, the cell wall and mitochondria. Moreover, this 

genome-wide analysis identified a number of gene products with unknown function which 

will be subjected to detailed molecular analysis addressing PtdEtn homeostasis in yeast cells. 

Figure 1: 

Three-dimensional reconstruction of 

yeast cells grown on oleic acid. 

Transmission electron micrographs of 

ultrathin sections (A, C; bar = 1µm) 

and corresponding 3D reconstructions 

of serial sections (B, D) of a 

chemically fixed wild type cell are 

shown. Cells shown in A and B were 

grown on YPD, and cells shown in C 

and D were grown on oleic acid to 

induce formation of peroxisomes. CW 

cell wall; ER endoplasmic reticulum; 

LP lipid particle; M mitochondrion; N 

nucleus; V vacuole; PX peroxisomes. 

By courtesy of G. Zellnig, KFUG. 

To understand PtdEtn homeostasis in some more detail, we performed experiments defining 

traffic routes of PtdEtn within the yeast cell. For these investigations we have chosen two 

subcellular fractions as destinations for PtdEtn translocation, namely peroxisomes and the 

plasma membrane. These two membranes have in common their lack of capacity to 

synthesize PtdEtn. We employed yeast mutants bearing defects in the different pathways of 

PtdEtn synthesis, which are distributed among mitochondria, endoplasmic reticulum and 

Golgi. These experiments demonstrated that PtdEtn formed in all three organelles can be 

supplied to peroxisomes and to the plasma membrane. The fatty acid composition of 

peroxisomal and plasma membrane phospholipids was mostly influenced by the available 

pool of total species synthesized, although a certain balancing effect was observed regarding 

the assembly of PtdEtn species into the two target compartments. We assume that the 

phospholipid composition of peroxisomes and the plasma membrane is mainly affected by 

the synthesis of the respective components and subject to equilibrium, and to a lesser extent 

affected by specific transport and assembly processes. Organelle association and membrane 

contact between organelles may be a relevant mechanism for lipid transport between 

20

organelles, but components governing this process and related events have to be studied in 

future investigations. 

Growth of yeast on oleic acid not only induces formation of peroxisomes, but also leads to an 

intense synthesis of triacylglycerols which are stored in lipid particles. The link between 

neutral lipid metabolism and peroxisome proliferation is subject to current investigations 

with emphasis on the role of enzymes involved. These studied are related to the possible 

lipotoxic effect of fatty acids in yeast. 

Neutral lipid storage in lipid particles and mobilization 

Yeast cells have the ability to store neutral lipids TAG (triacylglycerols) and SE (steryl esters) 

in subcellular structures named lipid particles/droplets. Upon requirement, TAG and SE can 

be mobilized and serve as building blocks for membrane biosynthesis. In an ongoing project 

in our laboratory, we investigate and characterize enzymatic steps which lead to the storage of 

TAG and SE and at the same time to the biogenesis of lipid particles. Moreover, we study 

processes involved in the mobilization of TAG and SE depots. 

Prerequisite for the understanding of LP function and structure is elucidation of their 

molecular equipment. For this purpose, we performed conventional analysis and mass 

spectrometric analysis of lipids (TAG, STE, phospholipids), but also of proteins which are 

present on the surface of LP. These analyses were carried out with LP from cells grown on 

glucose or oleic acid. Results obtained by these methods revealed marked differences in the 

lipidome, but also in the proteome of LP isolated from yeast grown under different conditions. 

Moreover, proteome analysis of LP led to identification of several new putative LP proteins. 

In the yeast, the SE synthases Are1p and Are2p, and the TAG synthases Dga1p and Lro1p 

contribute to the formation of lipid depots. Triple mutants with only one of these four 

enzymes active were used to obtain detailed information about the coordinate process of 

neutral lipid synthesis and the specific contribution of each of the four acyltransferases to lipid 

particle biogenesis. All four triple mutants with only one of the gene products, Dga1p, Lro1p, 

Are1p or Are2p, active form lipid particles, although at different rate and lipid composition. 

Through these experiments we showed that TAG or SE alone are sufficient for lipid particle 

biogenesis. Biophysical methods revealed that individual neutral lipids strongly affected the 

internal structure of lipid particles. SE form several ordered shells below the surface 

phospholipid monolayer of lipid particles, whereas TAG are more or less randomly packed in 

the center of the droplets (Figure 2). We propose that this structural arrangement of neutral 

lipids in lipid particles may be important for their physiological role especially with respect to 

mobilization of TAG and SE reserves. 

~5 shells SE TAG-core 

TG 

d~3500 Å 

d~ 200- 

300nm 

Figure 2: Internal structure 

of yeast lipid particles. 

A core of TAG is surrounded 

by several shells 

of SE. The surface of lipid 

particles consists of a 

phospholipid monolayer 

with proteins embedded 

21

Similar to TAG and SE synthesizing enzymes, proteins involved in degradation of neutral 

lipids occur in redundancy. Three TAG lipases named Tgl3p, Tgl4p and Tgl5p, and three SE 

hydrolases named Yeh1p, Yeh2p and Tgl1p were identified in the yeast. Recently, we studied 

enzymatic properties of the three SE hydrolytic enzymes in some detail. Our investigations 

demonstrated that each SE hydrolase exhibits certain substrate specificity. Using single, 

double and triple deletion mutants and strains overexpressing the three SE hydrolases, we 

showed the contribution of Tgl1p, Yeh1p and Yeh2p, respectively, to the mobilization of SE 

from their site of storage, the lipid particles. We also demonstrated that sterol intermediates 

stored as SE are readily mobilized through SE hydrolases, recycled to the sterol biosynthetic 

pathway and converted to the final product, ergosterol. Finally, we propose that defects in SE 

cleavage affect sterol biosynthesis in a type of feedback regulation. Conclusively, SE storage 

and mobilization although being dispensable for yeast viability under laboratory conditions 

contribute markedly to sterol homeostasis in this microorganism. 

Previous work from our laboratory had demonstrated that deletion of TGL3, the major yeast 

TAG lipase located to lipid particles, resulted in a decreased mobilization of triacylglycerols 

(TAG) from the lipid particles. TAG stored in a tgl3∆ mutant contained slightly increased 

amounts of 22:0 and 26:0 very long chain fatty acids (VLCFAs). These VLCFAs are 

indispensable for sphingolipid biosynthesis and crucial for raft association in the yeast. To 

understand the possible role of TAG lipolysis in sphingolipid metabolism, we carried out 

radiolabeling experiments with sphingolipid precursors in wild type and tgl mutants. These 

results indicated that the mutants have a significantly reduced rate of sphingolipid 

biosynthesis and an increased rate of sphingolipid turnover compared to wild type. 

Competition experiments with labeled palmitic acid (16:0) and non-radiolabeled cerotic acid 

(26:0) demonstrated that the 26:0 fatty acid competed with the 16:0 fatty acid during labeling 

of sphingolipids. In summary, we hypothesize that stored TAG can be a fatty acid donor for 

sphingolipid formation and TAG lipases contribute to this pathway in the yeast. 

Another potential non-polar storage lipid is squalene. This component belongs to the group of 

isoprenoids and is precursor for the synthesis of sterols, steroids and ubiquinons. It has 

become popular for biotechnological purposes but its isolation from natural sources, e.g., 

shark liver oil, is rather expensive and questionable from the ecological viewpoint. Therefore, 

alternative sources to isolate this compound were suggested among them the yeast 

Saccharomyces cerevisiae. In the yeast, the amount of squalene can be increased by varying 

culture conditions or by genetic manipulation. As an example, in strains deleted of HEM1 

squalene accumulates and is stored mainly in lipid particles. Interestingly, a heme deficient 

dga1∆lro1∆are1∆are2∆ quadruple mutant (Qhem1∆) which is devoid of the classical storage 

lipids, TAG and SE, accumulates substantial amounts of squalene in cellular membranes, 

especially in microsomes and the plasma membrane. The fact that Qhem1∆ does not form 

lipid particles suggests that biogenesis of these storage particles cannot be initiated by 

squalene which is synthesized in the ER similar to TAG and SE. This result also indicates that 

squalene accumulation, at least under the conditions tested, is not lipotoxic. Finally, our 

experiments demonstrate that squalene incorporated into organelle membranes does not 

compromise cellular function. In summary, subcellular distribution of squalene can be 

regarded as an example for highly flexible lipid storage in yeast. 

Lipids of Pichia pastoris organelles 

The yeast Pichia pastoris is an important experimental system for heterologous expression of 

proteins. Nevertheless, surprisingly little is known about organelles of this microorganism. 

For this reason, we started a systematic biochemical and cell biological study to establish 

22

standardized methods of Pichia pastoris organelle isolation and characterization. The 

expertise of our laboratory in cell fractionation and organelle isolation from the yeast 

Saccharomyces cerevisiae has been developed through a number of investigations in the past. 

Some of the methods applied for Saccharomyces cerevisiae can be used for Pichia pastoris 

cell fractionation, whereas others cannot or need to be adapted. Recent work focused on the 

biochemical characterization of mitochondrial membranes and the plasma membrane from 

Pichia pastoris grown on different carbon sources. The major aim of this project was the 

qualitative and quantitative analysis of phospholipids, fatty acids and sterols from both types 

of membranes; when Pichia pastoris was grown on different carbon sources. 

Recently, we started to study selected lipid synthesizing enzymes from Pichia pastoris with 

emphasis on the phosphatidylethanolamine (PE) biosynthetic machinery. We identified two 

Pichia pastoris PS decarboxylases (PSD) encoded by genes homologous to PSD1 and PSD2 

from Saccharomyces cerevisiae (Figure 3). Using Pichia pastoris psd1 and psd2 mutants we 

investigated the role of these two gene products in PE synthesis and the effect of their 

deletions on cell growth. Mitochondrial Psd1p is the major enzyme for PE synthesis in Pichia 

pastoris. Deletion of PSD1 caused a loss of PSD activity in mitochondria and a severe growth 

defect on minimal media which was partially cured by supplementation with ethanolamine. 

This result indicated that the CDP-ethanolamine but not the Psd2p pathway provided 

sufficient PE for cell viability in a psd1 background. Under these conditions, however, cellular 

and mitochondrial PE levels were dramatically reduced. Fatty acid analysis from wild type, 

psd1 and psd2 demonstrated the selectivity of both PSDs in vivo for the synthesis of 

unsaturated PE species. Short chain saturated (16:0) PE species were preferentially imported 

into mitochondria irrespective of psd1 or psd2 deletions. Thus, biosynthesis and subcellular 

distribution contribute to PE homeostasis in mitochondria of Pichia pastoris. 

. 

Figure 3 

Phylogenetic tree of PSD sequences from different organisms. The tree was constructed using 

the neighbor joining (NJ) method of the CLUSTALW program. 

23

Diploma Thesis completed 

Susanne Horvath: Phosphatidylserine Decarboxylase 1 from the Yeast Saccharomyces 

cerevisiae 

In the yeast Saccharomyces cerevisiae, phosphatidylethanolamine is synthesized via 4 

different pathways including phosphatidylserine decarboxylation by Psd1p and Psd2p, the 

CDP-ethanolamine pathway and the lysophospholipid acylation pathway (Ale1p). The PSD1 

proenzyme is composed of a mitochondrial targeting sequence, a ß-subunit, a cleavage site 

(LGST motif) and an α-subunit. To study the contribution of the α-subunit to posttranslational 

processing and enzymatic activation, mutants which lack the α-subunit, namely PSD1(-α)- 

GFP and PSD1(-α), were constructed. Both mutant strains were cloned into a psd2 

background and used for phenotype studies. Analysis of the growth phenotype of PSD1(-α)- 

GFP psd2 showed similarity to growth behaviour of psd1 psd2 which revealed an 

ethanolamine auxotrophy on minimal media containing lactate or glucose. In contrast, cells 

from PSD1(-α) psd2 were not able to grow on nonfermentable carbon sources such as glycerol 

and lactate even if ethanolamine was supplemented. Analysis of mitochondrial phospholipid 

patterns of the strains PSD1(-α)-GFP, PSD1(-α), PSD1(-α)-GFP psd2 and PSD1(-α) psd2 

exhibited a decreased level of phosphatidylethanolamine similar to psd1 and psd1 psd2 

mutants. This result suggests that the PSD1 proenzyme in yeast undergoes similar 

posttranslational processing steps as bacterial and mammalian PSD where a pyruvoyl 

prosthetic group at the amino terminus of the a-subunit is formed which is then part of the 

catalytic centre. Moreover, possible links between phosphatidylethanolamine and 

triacylglycerol metabolism were investigated. Therefore, triacylglycerol levels of mutant 

strains which bear defects in the four phosphatidylethanolamine biosynthetic pathways, 

namely psd1, psd2, psd1 psd2, cki1 dpl1 eki1 and ale1 were analyzed and compared to the 

wild type BY4741. On minimal lactate medium supplemented with ethanolamine, deletion of 

PSD1 led to decreasing triacylglycerol levels whereas in ale1 and cki1 dpl1 eki1 increased 

levels of triacylglycerol were observed, and psd2 showed a similar triacylglycerol level to wild 

type. These data suggest that phosphatidylethanolamine synthesis by Psd1p is directly linked 

to triacylglycerol metabolism. 

Doctoral Theses completed 

Andrea Wagner: Neutral lipid storage and mobilization in the yeast Saccharomyces 

cerevisiae 

In all types of eukaryotic cells, neutral lipids are stored as energy reserve and a source of 

building blocks needed for membrane formation. This is also true for the yeast S. cerevisiae 

which synthesizes triacylglycerols (TAG) and steryl esters (SE) as the most prominent storage 

lipids. These lipids are stored in so-called lipid particles (LP) which consist of a hydrophobic 

core of neutral lipids surrounded by a phospholipid monolayer with a small amount of 

proteins embedded. This thesis dealt with the role of LP in neutral lipid metabolism of the 

yeast. The first important point of this study was identification and characterization of SE 

hydrolases, and the second aspect was a structural investigation of LP. In the first part of this 

work the two SE hydrolases, Yeh1p and Tgl1p, both localized on LP were characterized. The 

role of two enzymes with overlapping activities and slightly different substrate specificities is 

discussed. In the second part of this thesis, yeast strains mutated in the neutral lipid 

synthesizing enzymes Dga1p, Lro1p, Are1p and Are2p were used for biochemical und 

biophysical investigations. It was demonstrated that the four acyltransferases act 

independently of each other and are able to synthesize individually a sufficient amount of 

24

neutral lipids for functional LP. A model of yeast LP suggesting SE to form several ordered 

shells around a central core of randomly packed TAG is proposed. 

Irmgard Schuiki: Assembly of phospholipids into mitochondrial membranes 

Phospholipids are essential components for function and integrity of membranes. In the yeast 

Saccharomyces cerevisiae, phosphatidylethanolamine (PtdEtn) plays a specific and important 

role. Yeast PtdEtn can be formed by four different pathways, with the major route mediated 

by phosphatidylserine decarboxylase 1 (Psd1p) localized to the inner mitochondrial membrane 

(IMM). Assembly of Psd1p to the IMM is affected by deletion of OXA1 encoding an IMM 

protein translocase, which leads to decreased PtdEtn synthesis in mitochondria. The IMM 

protease Yme1p contributes substantially to the proteolytic turnover of Psd1p. To obtain a 

genome wide view of the role of Psd1p, DNA microarray analyses were performed. This 

analyses revealed involvement of PtdEtn formed by Psd1p in numerous cellular pathways. 

Subcellular distribution of PtdEtn was studied with emphasis on the supply of this 

phospholipid to the plasma membrane of yeast. It was assumed that specific lipid transport 

from the intracellular sites of synthesis, the endoplasmic reticulum, mitochondria and Golgi 

compartment, has to occur towards the cell periphery. These investigations showed that 

supply of PtdEtn to the cell periphery can be accomplished by all pathways of PtdEtn 

synthesis. However, there is a preference for PtdEtn produced by phosphatidylserine 

decarboxylase 2 (Psd2p). In summary, this Thesis underlined the importance of PtdEtn, not 

only for the structural integrity of cellular membranes, but also for complex physiological 

processes. 

Tamara Wriessnegger: Characterization of organelles from the methylotrophic yeast Pichia 

pastoris 

The methylotrophic yeast Pichia pastoris is widely used as efficient expression system for the 

production of heterologous proteins, but despite the extensive use in biotechnology hardly any 

information is available about cell biological and biochemical properties of Pichia pastoris 

organelles. This thesis provided first insight into the membrane composition of peroxisomes, 

mitochondria and the plasma membrane of Pichia pastoris. We described appropriate 

methods for the isolation of highly purified organelles, which are prerequisite for subsequent 

protein and lipid analyses. Peroxisome induction by methanol or oleic acid leads to the 

accumulation of proteins of alcohol oxidation or fatty acid beta-oxidation, respectively, in 

peroxisomes. Morphological changes of Pichia pastoris in response to the growth medium 

were investigated by electron microscopy. The lipid composition of peroxisomal membranes 

shows only minor differences in response to the used carbon source. The exception was the 

use of oleic acid, because this fatty acid became predominant in phospholipids from total cell 

and peroxisomal extracts. On either C-source, phosphatidylcholine (PtdCho) and phosphatidylethanolamine 

(PtdEtn) were the major peroxisomal phospholipids. In mitochondria, a 

characteristic feature is the higher phospholipid to protein ratio and the lower ergosterol to 

phospholipid ratio of the outer membrane compared to the inner membrane. Variation of the 

C-source, however, leads to marked changes of the fatty acid pattern both in total and 

mitochondrial membranes. The inner mitochondrial membrane is also enriched in cardiolipin, 

whereas the outer membrane contains a high amount of phosphatidylinositol. Our study shows 

that PtdEtn synthesis by phosphatidylserine decarboxylase 1 (Psd1p) is mainly localized to 

mitochondria and Psd1p is responsible for the major PSD activity in Pichia pastoris. Defects 

in the enzyme lead to disability of the cells to grow on minimal glucose media without 

supplementation of ethanolamine. Psd2p, the second PSD enzyme, is not able to compensate 

25

for the loss of Psd1p, whereas the CDP-ethanolamine branch of the Kennedy pathway 

achieves major importance in PtdEtn synthesis of Pichia pastoris. Finally, we show that the 

lipid composition of the plasma membrane is very flexible and highly depends on the used C- 

source. Especially methanol and oleic acid lead to a change in plasma membrane lipid 

composition which may have a strong impact on membrane structure and fluidity. 

International Cooperations 

J. Cregg, Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA 

I. Hapala, Slovak Academy of Sciences, Institute of Animal Biochemistry and Genetics, 

Ivanka pri Dunaji, Slovak Republic 

I. Feussner, Plant Biochemistry, Albrecht-von-Haller-Institute of Plant Sciences, Georg- 

August University Göttingen, Germany 

R. Erdmann, Institute of Physiological Chemistry, Ruhr-University Bochum, Germany 

T. Höfken, Institute of Biochemistry, Christian Albrecht University, Kiel, Germany 

M. Karas, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University, 

Frankfurt, Germany 

R. Rajasekharan, Department of Biochemistry, Indian Institute of Science, Bangalore, India 

A. S. Sandelius, Department of Plant and Environmental Sciences, University of Gothenburg, 

Sweden 

Research Projects 

FWF 17321: Phosphatidylethanolamine of yeast mitochondria 

FWF 18857: Neutral lipid storage and mobilization in the yeast Saccharomyces cerevisiae 

FWF L-178 (Translational research): Characterization of organelles from the yeast Pichia 

pastoris 

FWF PhD Program: Molecular Enzymology 

Invited Lectures 

1) G. Daum 

Lipid storage and mobilization in the yeast 

Fluxome Company, Lyngby, Denmark, 16 October 2008 

2) G. Daum 

Lipid storage and mobilization in the yeast 

Croatian Biochemical Society, Osijek, Croatia, 17-20 September 2008 

Publications 

1) Czabany, T., Wagner, A., Zweytick, D., Lohner, K., Leitner, E., Ingolic, E. and Daum, 

G.: 

Structural and biochemical properties of lipid particles from the yeast Saccharomyces 

cerevisiae. 

J. Biol. Chem. 2008, 283:17065-17074. 

26

2) Rajakumari, S., Grillitsch, K. and Daum, G.: 

Synthesis and turnover of non-polar lipids in yeast 

Prog. Lipid Res. 2008,47:157-171. 

3) Schuiki, I., and Daum, G.: 

Phosphatidylserine decarboxylases, key enzymes of lipid metabolism 

IUBMB Life 2009 in press. 

4) Wagner, A., Grillitsch, K., Leitner, E. and Daum, G.: 

Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae. 

Biochim. Biophys. Acta. 2009 [Epub ahead of print]. 

5) Wriessnegger, T., Leitner, E., Belegratis, M. R., Ingolic, E. and Daum, G.: 

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris. 

Biochim. Biophys. Acta. 2009 [Epub ahead of print]. 

6) Connerth. M., Grillitsch, K., Köfeler, H. and Daum, G.: 

Analysis of Lipid Particles from Yeast 

Lipidomics, Humana Press, D. Armstrong ed. (2009) in press. 

Award 

Tibor Czabany received a FEBS Short term Fellowship for a stay in the laboratory of Prof. I. 

Feussner, Plant Biochemistry, Albrecht-von-Haller-Institute of Plant Sciences, Georg-August 

University Göttingen, Germany 

27

Molecular Biology Group 

Karin Athenstaedt 

Triacylglycerol synthesis in the oleaginous yeast Yarrowia lipolytica 

In the oleaginous yeast Yarrowia lipolytica mainly triacylglycerols (TAG) and only minor 

quantities of steryl esters accumulate as storage lipids. Like in all eukaryotic cells these 

neutral lipids are sequestered from the cytosolic environment in so-called lipid particles. The 

structure of these storage compartments of neutral lipids is rather simple. TAG and steryl 

esters are forming a hydrophobic core which is surrounded by a phospholipid monolayer with 

few proteins embedded. Under specific growth conditions, e.g., when industrial fats or 

glycerol are used as a carbon source, the amount of TAG can be increased up to 40% of cell 

dry weight. Microscopic inspection of cells grown under such conditions reveals that nearly 

the entire cell is filled with lipid particles. This outstanding capacity of Yarrowia lipolytica to 

accumulate neutral lipids (mainly TAG) leads to its application in biotechnological processes 

such as single cell oil production or production of nutrients enriched in essential fatty acids 

which can serve as nutritional complements. However, Yarrowia lipolytica may also serve as 

a model organism to study lipid turnover in adipocytes, since not only the ability to store 

excessive amounts of TAG in lipid particles but also the composition of this cell compartment 

resembles adipocytes of higher eukaryotes. Despite these potentials of Yarrowia lipolytica 

information about TAG (lipid) metabolism in this yeast is rather limited. Thus, recently we 

started to investigate the proteome of Yarrowia lipolytica for proteins involved in TAG 

synthesis. Computational analyses revealed two candidate gene-products of the oleaginous 

yeast homologous to TAG synthases of other organisms. A decreased amount of TAG in 

mutant cells defective in these candidate genes already pinpointed to a function of these 

polypeptides in TAG formation. To investigate whether these candidate genes encode true 

TAG synthases these genes were heterologously expressed in cells of a Saccharomyces 

cerevisiae mutant defective in neutral lipid synthesis and as a consequence lacking lipid 

particles. 

A 

C 

B 

D 

Figure 1: Restoration of lipid particle 

formation upon heterologous 

expression of Yarrowia lipolytica TAG 

synthase candidates in mutant cells of 

the budding yeast Saccharomyces 

cerevisiae lacking lipid particles. 

Upper panels: Positive (A; wild-type) 

and negative (B; mutant) control of 

Saccharomyces cerevisiae cells bearing 

empty plasmids. 

Lower panels: Saccharomyces 

cerevisiae mutant cells transformed 

with plasmids bearing either of the 

respective candidate genes of Yarrowia 

lipolytica (C, D). 

Arrows point to lipid particles. 

Size bar: 10 µm. 

28

Fluorescent microscopic inspection and lipid analyses of the respective mutants clearly 

demonstrated that these Yarrowia lipolytica genes encode true TAG synthases. Currently, the 

characteristics of these two TAG synthases of the oleaginous yeast are under investigation. 

Is Nte1p of Saccharomyces cerevisiae a true member of the NTE1-family? 

Neuropathy target esterase (NTE) was originally identified as the target of organophosphorous 

esters (OP) present in many pesticides as well as warfare agents. Toxifications with OPs are 

causing delayed neuropathy in man and some other vertebrates. The syndrome of the 

organophosphorous-induced delayed neuropathy (OPIDN) is characterised by paralysis of the 

lower limbs and degeneration of long axons in the spinal cord. These effects are common 

responses of neurons in metabolic disorders (e.g., diabetes), toxic states and advanced age. 

Neither NTE nor its homologs in other organisms are closely related to any other esterase or 

protease. The importance of NTE can be seen by the fact that mice lacking this protein die as 

early embryos, and inactivation of SWS (swiss cheese), the homolog of NTE in the fruit fly 

Drosophila melanogaster, leads to a progressive degeneration of the nervous system, which 

can be observed as big “holes” in the brain of the fly. 

Figure 2: “Holes” in the brain of a sws-mutant of the fruit fly Drosophila melanogaster. First 

“holes” in the brain of a sws-mutant can be already observed after 5 days (B). The number of 

“holes” increases progressively with age (20d; C). In contrast, no “holes” can be observed in 

wild-type brains after 20 days (A). 

Heterologous expression of the murine NTE can completely substitute SWS in Drosophila, 

demonstrating the functional conservation of this protein. A homolog of NTE is also present 

in the yeast Saccharomyces cerevisiae encoded by NTE1. In contrast to higher eukaryotes, 

deletion of this protein in the model organism yeast does not result in a phenotype under 

several standard conditions tested. However, due to the presence of this polypeptide in a 

single cell organism, NTE has to be involved in a more general context as in development, 

neural survival and brain integrity. All efforts to identify the biological function, substrate, 

and interaction partners of NTE in higher eukaryotes have failed so far. In contrast, Nte1p of 

the yeast has been reported to exhibit phospholipase B activity, thus indicating a similar 

function of the respective counterparts of higher eukaryotes. In this project, we are 

investigating whether Nte1p of Saccharomyces cerevisiae is a true member of the NTE 

protein family and thus suitable as a model for unravelling the biological function of the NTE 

protein family. In vitro, the functional conservation between NTE of higher eukaryotes and 

Nte1p of the yeast has already been demonstrated. But is this also the case in vivo? For 

answering this question, I expressed NTE1 of the yeast heterologously in the fruit fly 

Drosophila melanogaster. A functional compensations would be indicated by reversion of the 

phenotype of an sws mutant fly, i.e., observable as a decrease in the size and / or number of 

“holes” in the brain of Drosophila. Several different mutant lines were constructed expressing 

the yeast NTE1 to different extent and in different tissues. Subsequently, the brains of these 

29

mutants were analyzed by fluorescent microscopy for the presence of “holes”. Any result from 

full reversion of the phenotype to no effect has been considered, however, the phenotype was 

even worse in the transfectants, showing a higher number of “holes”. In addition, the life span 

of the sws-mutants expressing NTE1 of yeast was significantly reduced compared to control 

flies. This result indicates that there is no functional conservation among the NTE protein 

family in vivo. However, my searches for an explanation what caused this rather striking effect 

provided some evidence that Nte1p interacts with enzymes of the lipid metabolism and forms 

a protein complex with at least one more polypeptide. Preliminary results indicated that this is 

also true for Sws of the fruit fly. Furthermore, mutations in certain lipid synthesizing 

pathways led to smaller and a fewer number of holes in the brain of a sws-mutant fly, thus 

indicating that the phenotype of the sws mutant is caused by major changes in the lipid 

pattern. Analysis of the lipid pattern of the respective mutant flies will reveal which lipid 

parameters are required for normal development of the nervous system, and will provide 

valuable information for potential targets to compensate effects caused by neurons in 

metabolic disorders (e.g., diabetes), toxic states and advanced age. 

International Cooperations 

T. Chardot and J.-M. Nicaud, Institute National de la Recherche Agronomique, UMR Chimie 

Biologique, Thiverval Grignon, France 

D. Kretzschmar, Oregon Health and Sciences University, Portland, Oregon, USA 

P. Glynn, Leicester University, Leicester, UK 

Publications 

1) Athenstaedt, K.: 

Neutral lipids in yeast: synthesis, storage and degradation. 

In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N. 

Timmis) Springer-Heidelberg, 2008 in press. 


Players in the neutral lipid game – proteins involved in neutral lipid metabolism in yeast. 




Isolation and characterization of lipid particles of yeast. 




Neutral lipid metabolism in yeast as a template for biomedical research. 



30

Biophysical chemistry group 

Group leader: Albin Hermetter 

Post-docs: Heidrun Susani-Etzerodt, Jo-Anne Rasmussen, Heidemarie Ehammer 

Graduate students: Jessica Schatte, Maria Morak, Maximilian Schicher, Ute Stemmer, Tomas 

Bobula, Zsuzsanna Dunai, Daniel Koller, Claudia Ramprecht, Lingaraju Marlingapla 

Halasiddappa 

Undergraduate students: Martina Geier 

Technicians: Elfriede Zenzmaier 


Our research deals with the role of glycero(phospho)lipids and lipid modifying enzymes as 

components of membranes and lipoproteins, as mediators in cellular (patho)biochemistry, and 

their application as analytical tools in enzyme technology. Fluorescence spectroscopy is used 

as a main technique to investigate the behaviour of these biomolecules in the respective 

supramolecular systems. 

Section 1 of the following report summarizes our studies on lipid oxidation, the effects of 

oxidized lipids on intracellular signalling, and the inhibition of these processes by synthetic 

and natural antioxidants. 

Section 2 describes the development of fluorescence and chip technology for functional 

proteomic analysis of lipolytic enzymes in microbial, animal and human cells. 

1. Lipid oxidation and atherosclerosis – Lipotoxicity 

1.1. Interaction of oxidized phospholipids with vascular cells 

Sphingomyelin 

acid 

Sphingomyelinase 

Oxidized - 

Phospholipids 

O 

H 

O 

O 

O 

O 

O 

O 

O 

O 

O 

O P O 

OH 

O 

O 

O 

O P O 

OH 

+ 

N 

N + 

Ceramide 

JNK 

p38 MAPK 

Caspase 3 

ERK 

AKT/PKB 

NFkB 

PROLIFERATION 

SURVIVAL 

Interactions of oxidized lipoproteins with 

the cells of the arterial wall induce and 

influence the progress of atherosclerosis. 

Accumulation of foam cells originating 

from macrophages and excessive intimal 

growth of vascular smooth muscle cells 

(SMC) alternating with focal massive cell 

death are characteristics typical of the atherosclerotic lesion. These phenomena are largely 

mediated by the oxidized phospholipid components of the modified particles that are 

generated under the conditions of oxidative stress. In the framework of the research 

consortium LIPOTOX, we investigate the “Toxicity of oxidized phospholipids in 

macrophages”. This study aims at identifying the molecular and cellular effects of an 

important subfamily of the oxidized phospholipids containing long hydrocarbon chains in 

position sn-1 and short polar acyl residues in position sn-2 of the glycerol backbone. The 

respective compounds trigger an intracellular signaling network including sphingomyelinases, 

(MAP) kinases and transcription factors thereby inducing proliferation or apoptosis of 

vascular cells. Both phenomena largely depend on the specific action of sphingolipid 

31

mediators that are acutely formed upon cell stimulation by the oxidized lipids. Fluorescence 

microscopic studies on labeled lipid analogs revealed that the short-chain 

phosphatidylcholines are easily transferred from the aqueous phase into the cell plasma 

membrane and eventually spread throughout the cells. Under these circumstances, the biologi- 

cally active compounds are very likely to interfere 

directly with various signaling components inside 

the cells, which finally decide about cell growth or 

death. Investigations are being performed on the 

levels of the lipidome, the apparent enzyme 

activities, the proteome an the transcriptome to find 

the primary molecular targets and their downstream 

elements that are the key compnents of lipid-induced 

cell death. 

1.2. Oxidative stress and antioxidants 

Antioxidants protect biomolecules against 

oxidative stress and thus, may prevent its 

pathological consequences. Specific fluorescent 

markers have been established for high-throughput 

screening of lipid and protein oxidation and its 

inhibition by natural and synthetic antioxidants. 

These methods can also be used for the 

determination of antioxidant capacities of 

biological fluids (e.g. serum) and edible oils. A 

prominent example is pumpkin seed oil which 

contains a variety of antioxidants (e.g. tocopherols 

and phenolic substances) and other secondary plant 

components that possess useful biological 

properties. 

2. Functional proteomic analysis of lipolytic enzymes 

The functional properties of lipases and esterases are the subject of our studies in the 

framework of two joint research programs (GOLD-Genomics of Lipid-associated Disorders at 

KFU Graz and the Research center Applied Biocatalysis at TU Graz). Lipolytic enzymes 

catalyze intra- and extracellular lipid degradation. Dysfunctions in lipid metabolism may lead 

to various diseases including obesity, diabetes or atherosclerosis. In chemistry, lipases and 

esterases are important biocatalysts for (stereo)selective reactions on synthetic and natural 

substrates leading to defined products for pharmaceutical or agrochemical use. 

2.1. Fluorescent suicide inhibitors for in-gel analysis of lipases and esterases 

Fluorescent suicide inhibitors have been developed as activity recognition probes (ARPs) for 

qualitative and quantitative analysis of active lipolytic enzymes in complex biological samples 

(industrial enzyme preparations, serum, cells, tissues). Since inhibitor binding to the active 

32

sites of lipases and esterases is specific and stoichiometric, accurate information can be 

obtained about the type of enzyme and the moles of active protein (active sites) in 

electrophoretically pure or heterogeneous enzyme preparations. 

Labelling of enzymes with an ARP specific for serine hydrolases 

BG 

Inactive 

Inactive 

RG NBD 

RG: Reacti v e p h osphonate gr o up, 

irreversibly inhibits the catalytic serine 

Active 

BG 

BG: Binding group: is specifically recognized 

by the active enzyme 

Inactive 

Inactive 

Inhibited 

RG 

NBD 

NBD : fluorescent tag 

λ : 488 nm; λ : 540 nm 

ex 

em 

Fluorescent inhibitors are currently applied to proteomic analysis of the lipolytic enzymes in 

human and animal cells. These studies are performed in the framework of the joint project 

GOLD (Genomics of Lipid-associated Disorders/ coordinated by KFU Graz) which aims at 

discovering novel genes, processes and pathways that regulate lipid homeostasis in humans, 

mice and yeast. This is one of the projects in the field of functional genomics (GEN-AU, 

GENome research in AUstria) funded by the Austrian Federal Ministry for Education, Science 

and Culture (bm:bwk). 

Activity-based enzyme stain 

Total protein stain 

The lipolytic proteome of mouse 

adipose tissue 

Proteins of brown adipose tissue from fasted wild-type mice were labelled with a fluorescent 

lipase inhibitor followed by 2-D electrophoretic separation. The fluorescently tagged enzymes 

were detected with a laser scanner. A total protein stain (Sypro rubyTM) is shown as a 

control. Fluorescent spots were cut out and after tryptic in gel-digest, the isolated peptides 

were analyzed by nanoHPLC-MS/MS. The lipolytic proteomes of various mouse tissues, 

human liver and cultured human adipocytes have been determined using this method. The 

activities of new enzyme candidates on physiologically relevant lipid substrates are currently 

being identified. 

33

2.2. Protein microarrays – Enzyme chips 

In the framework of the Kplus Research Centre 

Applied Biocatalysis (TU Graz), we have 

developed "biochips" representing ordered 

microarrays of biospecific ligands for the detection 

and quantification of lipolytic enzymes in 

biological samples. In addition, microarrays of 

enzymes are generated to determine protein 

affinities for substrate analogs. The development 

of these tools basically requires three steps, namely 

specific loading of activated supports with 

bioaffinity ligands, measurement of lipase binding 

(e.g. by fluorescence techniques), and application 

Angewandte 

Biokatalyse 

Kompetenzzentrum GmbH 

of the bio chips to the investigation of real analytical problems. These systems will be used 

for the screening of lipases with specific substrate acceptance. 

Diploma thesis completed: 

Martina Geier: Functional screening of lipolytic enzymes in mouse tissues. 

In the postgenomic era, the focus of research is on the identification of the gene products and 

on the assignment of their cellular and physiological functions. In this thesis, various mouse 

tissues were profiled for esterolytic and lipolytic proteins using biotinylated 

p-nitrophenylphosphonates with distinct selectivities. The functional screens showed that the 

complexity of the lipolytic proteome varies notably between the examined tissues. A 

comparison of the data sets obtained with the novel biotinylated probes on the one hand and 

established fluorescent inhibitors on the other hand revealed that information from both types 

of inhibitors is complementary. The functional screen of mouse tissues revealed new potential 

esterase and lipase candidates: a hydrolase domain containing protein 10 (Abhd10), 

lysophospholipase 3 (LPL) and an unknown protein product (UPP) were found in white 

adipose tissue. These proteins were cloned, transiently over-expressed in COS-7 cells and 

examined with regard to their substrate preferences. Finally, a set of matched fluorescence 

inhibitors for detection of lipases was synthesized, characterized and applied to differential 

activity-based analysis (DABGE) of isolated enzymes. 

Doctoral thesis completed: 

Jessica Schatte: Molecular identification of lipases in liver and bile 

This work aimed at the molecular identification of lipolytic enzymes in liver and bile by a 

functional proteomic approach. Bile has been thought to be mainly involved in lipid 

metabolism by emulsifying lipids in the upper intestine. In addition, bile expresses lipolytic 

activity but no respective enzymes have been identified at the molecular level so far. It was 

the aim of the here presented study to identify these proteins. We determined the lipolytic 

activity of murine bile with structurally defined glycerolipids. Additionally, we identified 

several lipolytically and esterolytically active enzymes using active site-directed fluorescent 

inhibitors followed by two-dimensional gel electrophoresis and mass spectroscopy. Two of 

the identified lipases, Triacylglycerol Hydrolase (TGH) and Carboxylesterase ML1 (CES 

34

ML1) were chosen for detailed analysis. Both enzymes hydrolised triacylglycerols in vitro. In 

contrast to TGH, CES ML1 also hydrolised diacylglycerols. Thus we assume that in addition 

to other lipases, CES ML1 and TGH account for a considerable share of the lipolytic activity 

of bile. In addition, we identified the lipolytic and esterolytic proteome of human liver and 

cultured human cells (HepG2), which are responsible for protein secretion into bile, using the 

same activity-based proteomics approach mentioned above. A quantitative comparison of 

liver tissue from different donors using differential activity-based gel electrophoresis 

(DABGE) showed that every individual expressed a distinct enzyme pattern. This work 

represents the first detailed study describing the lipolytic enzymes in human liver tissue and 

liver cells. The functional proteomic approach used therein can be expected to become a 

valuable tool for future investigations on liver lipid (patho) physiology 

International Cooperations: 

T. Hianik, Department of Biophysics and Chemical Physics, Comenius University, Bratislava, 

Slovakia 

E. Lacôte, Chargé de recherche CNRS, Université Pierre et Marie Curie, Paris, France 

C. Ferreri, I.S.O.F. - BioFreeRadicals , Consiglio Nazionale delle Ricerche, Bologna Italy 

C. Tringali, Dipartimento di Scienze Chimiche, Universita di Catania, Italy 

D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine; 

Cornell University, Ithaca, USA 

R. Yates, Department of Biochemistry and Molecular Biology, Faculty of Medicine, 

University of Calgary, Calgary, Canada, 

N. Faergeman, Department of Biochemisty and Molecular Biology, University of Southern 

Denmark, Odense, Denmark 

Research Projects: 

BM.W_F (Genomforschung in Österreich, GEN-AU): Functional and differential analysis of 

lipolytic enzymes by affinity tagging (GOLD II project-Genomics of Lipid-associated 

Disorders, coordinated by KFU Graz). 

FWF Special Research Program (SFB) LIPOTOX (coordinated by KFU Graz): Toxicity of 

oxidized phospholipids in macrophages. 

FWF Doctoral Program Molecular Enzymology: Functional enzyme analysis (coordinated by 

KFU Graz). 

TIG Research center Applied Biocatalysis (coordinated by TU Graz): Novel lipases with 

specific substrate acceptance. 

Invited lectures: 

1) Mechanisms of signaling by oxidized phospholipids in vascular cells. J. Heyrovský 

Institute of Physical Chemistry, Academy of Sciences of the Czech R., Czech Republic, 

September 26, 2008. 

2) Functional proteomic analysis of lipolytic enzymes in mammalian cells. Medical 

University of Innsbruck, Austria, June 5, 2008 

3) Apoptotic signalling of oxidized phospholipids in vascular cells. COST meeting 

“Bioradicals”, Bologna, Italy, April 11, 2008 

35

Publications: 

1) Deigner, H. P., Hermetter, A.: 

Oxidized phospholipids: Emerging mediators of pathophysiology 

Curr. Opin. Lipidol. 2008, 19:289-294. 

2) Birner-Gruenberger, R., Susani-Etzerodt, H., Kollroser, M., Rechberger, G., Hermetter, 

A.: 

Activity-based proteome mapping of lipases and esterases in murine liver 

Proteomics 2008, 8:3645-3656. 

3) Rasmussen, A., Hermetter, A.: 

Chemical synthesis of fluorescent glycero- and sphingolipids 

Progr. Lipid Res. 2008, 47:436-460. 

4) Steiner, A., Stütz ,A., Wrodnigg, T. M., Tarling, C. A., Withers, S. G., Hermetter, A., 

Schmidinger, H.: 

Glycosidase profiling with immobilized glycosidase inhibiting iminoalditols - A proofof-concept 

study 

Bioorg. Med. Chem. Lett. 2008, 18:1922-1925. 

5) Fruhwirth G. O., Hermetter A.: 

Production technology and characteristics of Styrian pumpkin seed oil 

Dossier “Virgin (cold-pressed) oils”, B. Matthäus, ed. 

Eur. J. Lipid Sci. Technol. 2008, 110:637-644. 

6) Chatilialoglu, C., Ferreri, C., Hermetter, A., Lacote, E., Mihaljevic, B., Nicolaides, A., 

Siafaka-Kapadai, A.: 

Lipidomics and free radical modifications of lipids 

Chimia 2008, 62:713-720. 

7) Fruhwirth, G. O., Hermetter, A.: 

Mediation of apoptosis by oxidized phospholipids 

Subcellular Biochemistry 2008, 49:351-367. 

8) Grimard, V., Massier, J., Richter, D., Schwudke, D., Kalaidzidis, Y., Fava, E., 

Hermetter, A., Thiele, C.: 

siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride 

homeostasis 

J. Lipid. Res. 2008, 49:2427-2440. 

9) Schicher, M., Polsinger, M., Hermetter, A., Prassl, R., Zimmer, A.: 

In vitro release of propofol and binding capacity with regard to 

plasma constituents 

Eur. J. Pharm. Biopharm. 2008, 70:882-888. 

10) Rhode, S., Grurl, R., Brameshuber, M., Hermetter, A., Schütz, G. J.: 

Plasma membrane fluidity affects transient immobilization of oxidized phospholipids in 

endocytotic sites for subsequent uptake. 

J. Biol. Chem., 2008, Epub ahead of print. 

36

11) Watschinger, K., Keller, M. A., Hermetter, A., Golderer, G., Werner-Felmayer, G., 

Werner, E. R. 

Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal 

ion and tetrahydrobiopterin dependence. 

Biol. Chem., 2008, Epub ahead of print. 

Patents: 

Optisch detektierbare (Alkylacyl-2-Aminodesoxy-glycero)-p- nitrophenyl-phosphonate. 

Europäische Patentschrift EP 1 603 927 B1 (20. 8. 2008) 

Erfinder: A. Hermetter und O. Oskolkova 

37

Lectures and Laboratory Courses 

LU aus Biochemie I 

5,33 LU Staff 

Biochemie II 1,5 VO Macheroux P 

LU aus Biochemie II 4 LU Staff 

Bachelorarbeit 1 SE Daum G 

Seminar zur Masterarbeit aus Biochemie und 

Molekularer Biomedizin 

2 SE Daum G, Hermetter A, 

Macheroux P 

Anleitung zu wissenschaftlichen Arbeiten aus Biochemie 2 SE Daum G, Hermetter A, 

und Molekularer Biomedizin 

Macheroux P 

Molekulare Physiologie 2 VO Macheroux P 

Projektlabor Bioanalytik 6 PR Daum G, Hermetter A, 

Macheroux P 

Molekulare Enzymologie I 3 PV Macheroux P 

GL Chemie (BMT) 2 VO Hermetter A 

Membran Biophysik 3 PV Lohner K 

Membran-Mimetik 1 VO Lohner K 

Biophysikalische Chemie 3 PV Laggner P 

Biophysikalische Chemie der Lipide 3 PV Hermetter A 

Zellbiologie der Lipide 3 PV Daum G 

Zellbiologie 1 1 SE Daum G 

Physik.Meth.i.d.Biochemie 2 LU Laggner P 

Physikalische Methoden in der Biochemie 1 VO Laggner P 

Biochemie I 

3,75 VO Macheroux P 

LU aus Molekularbiologie 3 LU Athenstaedt K 

Seminar zu den LU aus Molekularbiologie 1 SE Athenstaedt K 

Immunologische Methoden 2 VO Daum G 

Immunologische Methoden 2 LU Daum G 

Bachelorarbeit 1 SE Daum G 

Seminar zur Masterarbeit aus Biochemie und 

Molekularer Biomedizin 

2 SE Daum G, Hermetter A, 

Macheroux P 

Anleitung zu wissenschaftlichen Arbeiten aus Biochemie 2 SE Daum G, Hermetter A, 

und Molekularer Biomedizin 

Macheroux P 

Spezielle Kapitel der Biochemie 1 VO Daum G, Hermetter A, 

Macheroux P 

Fluoreszenztechnologie 2 VO Hermetter A 

Fluoreszenztechnologie 1,5 LU Hermetter A 

Molekulare Enzymologie 2 VO Gruber K, Macheroux 

P, Nidetzky B 

Projektlabor Bioanalytik 6 PR Daum G, Hermetter A, 

Macheroux P 

Molekulare Enzymologie II 3 PV Macheroux P 

GL Biochemie (BMT) 2 VO Macheroux P 

Membran Biophysik 3 PV Lohner K 

Biophysikalische Chemie 3 PV Laggner P 

Biophysikalische Chemie der Lipide 3 PV Hermetter A 

Zellbiologie der Lipide 3 PV Daum G 

Strukturanalyse in Biophysik u. Materialforschung 2 VO Laggner P 

Zellbiologie 2 1 SE Daum G 

Immunologische Methoden 1 VO Daum G 

38

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