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 

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

Dr. Sigrid Deller 

(Sigrid.deller@tugraz.at; Tel.: +43-(0)316-873-6955) 

Mag. rer. nat. Gernot Rauch 

(gernot.rauch@tugraz.at; Tel.: +43-(0)316-873-6454) 

Mag. rer. nat. Heidemarie Ehammer 

(h.ehammer@tugraz.at, Tel.: +43-(0)316-873-6463) 

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 

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

6464 

Anna Prem; anna.prem@tugraz.at; Tel.: 43-(0)316-873-6467 

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

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

Eva Pointner; eva.pointner@tugraz.at; Tel.: +43-(0)316-873-6452 

Leo Hofer (Workshop); Tel.: +43-(0)316-873-8431 or 8433 

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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 dryrot 

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. 

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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 

Technical University in this field (SFB Biocatalysis & Kplus). 

3

Highlights of 2007 

In April, Peter Macheroux’s group hosted a Cost B22 meeting on “Drug Target 

Characterisation” at Schloss Seggau, near Graz. This conference was dedicated to 

vitamin and cofactor biosynthesis in protozoan parasites. The meeting was held from the 

27 th to 29 th of April 2007 and was jointly organised by the members the EU-funded 

VITBIOMAL consortium. During the meeting, more than 30 experts from Europe, 

Australia and the United States discussed the potential to develop useful drugs against 

the biosynthetic pathways of vitamins and drugs in protozoan parasites that cause severe 

health problems in tropical regions, such as malaria. In spring 2007, the FWF approved 

a project in P. Macheroux’s group to investigate enzymes involved of nikkomycin 

biosynthesis. The aim of the three-year project is to unravel the enzymatic steps that 

lead from basic metabolites to aminohexuronic acid, which constitutes one of the 

building blocks of peptidyl nucleosides such as nikkomycins produced by some 

Streptomycetes. The project is a joint effort between P. Macheroux’s group and Prof. 

Gruber’s team at the Graz University and further strengthens the scientific interactions 

of NAWI Graz and the FWF-funded PhD program “Molecular Enzymology”. 

As in the years before, research in Günther Daum’s group was supported by several 

grants of the Austrian Science Fund (FWF). Teaching and administrative activities 

included appointment as a member of the Studienkommission Biomedical Engineering 

and chairman of the Doctoral School „Molecular Biochemistry and Biotechnology“ of 

the TU Graz. As research related activities G. Daum worked as a Board Member of the 

Austrian Science Fund (FWF), Associate Editor of FEMS Yeast Research, and Member 

of the Editorial Board of the Journal of Biological Chemistry. Moreover, he was elected 

Vice President of the Austrian Society of Biochemistry and Molecular Biology, and 

continued his activities as a Vice President of the International Conference on the 

Bioscience of Lipids (ICBL) and Chairman of the Yeast Lipid Conference (YLC). 

Moreover, the European Federation for the Science and Technology of Lipids (Euro Fed 

Lipid) appointed G. Daum organizer of the Euro Fed Lipid Conference in Graz, Austria, 

in 2009. In 2007, invitations to distinguished research conferences in Italy, Finland and 

the USA were a great honour and an excellent opportunity to meet colleagues from the 

same field of research and discuss problems of mutual interest. 

Albin Hermetter started a new project in the framework of the research consortium 

(SFB) LIPOTOX funded by the Austrian Science Fund (FWF). It is based on a 

functional genomic study and aims at identifying the molecular basis of “The toxicity of 

oxidized phospholipids in macrophages” which is supposed to play a key role in the 

development of atherosclerosis. Since last year, A Hermetter is an Austrian 

representative in the COST action CM0603 which is devoted to “Bioradicals”. His 

contribution to the scientific program comprises various aspects of lipid oxidation and 

its biological consequences. The involvement of A. Hermetter’s group in industrial 

research and the continuous development of novel fluorescence technology have led to 

several patents during the last few years. Consequently, our institute became the fourth 

most successful department of TU Graz in the area of IPR and patenting. 

4

The PhD program “Molecular Enzymology” has received much attention recently as 

it has attempted to set new standards for the PhD education in Graz and beyond. Thanks 

to the generous financial endowment by the FWF, both universities, the state of Styria 

and the city of Graz, this “Doktoratskolleg” has been very successful. In January 2008, 

an application for an extension of the PhD program has been evaluated by an 

international board of reviewers. At present, seven PhD students of the 

“Doktoratskolleg” are trained in our institute, all the more reason to keep our fingers 

crossed that an extension will be granted! 

5

Biochemistry group 

Group leader: Peter Macheroux 

Secretary: Annemarie Portschy 

Senior Research Scientist: Sigrid Deller 

PhD students: Asma Asghar (since December 1st), Alexandra Binter (since October 

1st), Heidi Ehammer, Karlheinz Flicker, Martina Neuwirth, Sonja Sollner, Gernot 

Rauch, Andreas Winkler 

Diploma students: Alexandra Binter (until September 30th), Katharina Fallmann, Daniel 

Koller, Sabrina Riedl, Markus Schober, Silvia Wallner 

Guest students: Sebastian Hofzumahaus (FH Jülich, Germany), Nina Jajcanin (Ruder 

Boscovic Institute, Zagreb, Croatia), 

Sokrates student: Lucie Lorkova, Institute of Chemical Technology, Prague, Czech 

Republic 

Technicians: Rosemarie Trenker-El-Toukhy (karenziert), Eva Maria Pointner 

(karenziert), Anna Prem (Karenzvertretung), Martin Puhl (Karenzvertretung) 

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 regiospecificity). 

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 sitedirected 

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. 

The methods established in our laboratory comprise 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- 

6

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: 

H 3 C O 

HO 

H 

N 

CH 3 

OH 

OCH 3 

O 2 H 2 O 2 

berberine bridge enzyme 

(flavin-dependent) 

H 3 C O 

HO 

H 

N 

CH 2 

OH 

OCH 3 

(S)-reticuline 

(S)-scoulerine 

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 investigate this 

reaction in order to unravel its chemical mechanism and to elucidate the threedimensional 

structure of the protein. This will allow a detailed analysis of the structurefunction 

relationships in the enzyme active site and will enable us to probe the 

mechanism by mutagenic analysis. 

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

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

the protein. 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 crystallise 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 can now initiate a site-directed 

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

of the enzyme (thesis project of Andreas Winkler; diploma project of Sabrina Riedl). 

7

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/bifunctionality 

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 

8

eceives 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). 

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: 

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 

9

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 and Asma Asghar; 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 suger 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 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 threedimensional 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). 

10

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 threedimensional 

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. 

These studies were complemented by localization studies of LOT6p in yeast, which 

confirmed the cytosolic occurrence of the protein (in collaboration with Prof. Daum, TU 

Graz). In order to further characterize the cellular role of LOT6p, we are now in the 

process to search for protein interaction partners (thesis project of Sonja Sollner and 

diploma project of Markus Schober). 

Interestingly, the bacterial homolog of LOT6p, YhdA, forms stable tetramers instead of 

dimers. Apparently, this higher oligomeric state gives rise to increased thermostability 

(Deller et al., 2006). Inspection of the three-dimensional structure led to the 

identification of four salt-bridges, which hold two dimers together (see figure). 

11

To investigate the importance of these contacts for the formation of tetramers, we have 

generated a K109L and D137L single as well as a K109L/D137L double mutant protein. 

Currently, we are characterizing these muteins with respect to oligomerisation, 

temperature stability and catalytic activity. With this approach we hope to contribute to 

the understanding of protein oligomerization in general (research project of Dr. Sigrid 

Deller and diploma project of Alexandra Binter). 

Diploma thesis completed 

Alexandra Binter: Characterization of Bacillus subtilis NADPH:FMN oxidoreductase 

YhdA in respect to oligomerization, activity and thermostability. 

YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, shows 

quinone reductase activiy and occurs as a tetramer. The two extended dimer surfaces are 

packed against each other by a 90º rotation of one dimer with respect to the other. This 

assembly is stabilized by the formation of four salt bridges between K109 and D137. To 

investigate the importance of those ion pair contacts, the following muteins were created 

and heterologously expressed in E. coli: K109L and D137 single muteins and 

K109L/D137L and K109/D137K double muteins. These muteins were characterized 

with respect to oligomerization, showing that all the muteins form dimers in solution. 

Only K109L/D137L is partially tetrameric in concentrated solutions and tetrameric in 

crystals. Consequently, the salt bridges are essential for stabilizing the dimer-dimer 

interface. It was shown that thermal denaturation of the dimeric muteins occurs at 

equally high temperatures as it does in the case of the wild-type protein. The activity as 

and NADPH:FMN oxidoreductase using oxygen and various quinones as substrates is 

reduced drastically compared to wild-type YhdA. Interestingly, the rate of the oxidative 

half reaction is similar to the wild-type, whereas the rate of the reductive half reaction is 

reduced significantly. Thus, it is concluded that binding of NADPH to the dimeric 

muteins is impaired. 

International cooperations 

M. Abramic, Ruder Boskovic Institute Zagreb, Croatia 

N. Amrhein and T. B. Fitzpatrick, Institute of Plant Sciences, ETH-Zürich, Switzerland 

S. Ghisla, Fachbereich Biologie, Universität Konstanz, Germany 

I. Jelesarov, Universität Zürich, Switzerland 

B. Kappes, Universitätklinikum 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: “Enzyme der Nikkomycinbiosynthesis” 

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

FWF P17471: “Mechanism of chorismate synthase” 

EU-STRP: “Vitamin biosynthesis as target for antimalarial therapy” (VitBioMal) 

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FWF-Doktoratskolleg “Molecular Enzymology” 

WTZ Austria-Croatia: “Structure-function studies on metallopeptidases involved in 

metabolism of biologically active peptides” 

Invited lectures 

1) Cost B22 WG2 conference on “Drug target characterization” at Schloss Seggau, 

Austria (27.04.2007): “Thermodynamic protein-protein 

interaction studies on PfPdx1 and PfPdx2 from Plasmodium falciparum”. 

2) Universität Novi Sad, Serbia (04.06.2007): “Flavoenzyme catalysis in 

biosynthetic reactions”. 

Publications 

1) Kommoju, P. R., Macheroux, P., and Ghisla, S.: 

Molecular cloning and expression of L-amino acid oxidase from the Malayan pit 

viper Calloselasma rhodostoma, Protein Expr Purific. 2007, 52:89-95. 

2) Sollner, S., Nebauer, R., Ehammer, H., Prem, A., Deller, S., Palfey, B. A., 

Daum, G., and Macheroux, P.: 

LOT6 from Saccharomyces cerevisiae is a FMN-dependent reductase with a 

potential role in quinone detoxification, The FEBS Journal 2007, 274:1328-1339. 

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

Mutagenic analysis of an invariant aspartate residue in chorismate synthase 

supports its role as an active site base, Biochemistry 2007, 46:3768-3774. 

4) Neuwirth, M., Flicker, K., Strohmeier, M., Tews, I., and Macheroux, P.: 

Thermodynamic characterization of the protein-protein interaction in the 

heteromeric Bacillus subtilis pyridoxal synthase, Biochemistry 2007, 46:5131- 

5139. 

5) Hall, M., Stueckler, C., Kroutil, W., Macheroux, P., and Faber, K.: 

Asymmetric bioreduction of activated alkenes using cloned 12-oxophytodienoate 

reductase isoenzymes OPR-1 and OPR-3 from Lycopersicon esculentum 

(tomato): A striking switch of stereopreference, Angew. Chem. Int. Ed. 2007, 

46:3934-3937. 

6) Pricelius, S., Held, C., Sollner, S., Deller, S., Murkovic, M., Ullrich, R., 

Hofrichter, M., Cavaco-Paulo, A., Macheroux, P., Guebitz, G. M.: 

Enzymatic reduction and oxidation of fibre-bound azo-compounds, Enzyme 

Microb. Techn. 2007, 40:1732-1738. 

7) Winkler, A., Kutchan, T. M., and Macheroux, P.: 

6-S-Cysteinylation of bi-covalently bound FAD in berberine bridge enzyme 

tunes the redox potential for optimal activity, J. Biol. Chem., 2007, 282:24437- 

24443. 

13

8) Ehammer, H., Rauch, G., Prem, A., Kappes, B., and Macheroux, P.: 

Conservation of NADPH utilization by chorismate synthase and its implications 

for the evolution of the shikimate pathway, Mol. Microbiol., 2007, 65:1249- 

1257. 

9) Fitzpatrick, T. B., Amrhein, A., Kappes, B., Macheroux, P., Tews, I., and 

Raschle, T.: 

Two independent routes of de novo vitamin B6 biosynthesis: Not that different 

after all, Biochem. J., 2007, 407:1-13. 

10) Flicker, K., Neuwirth, M., Strohmeier, M., Kappes, B., Tews, I., and Macheroux, 

P.: 

Structural and thermodynamic insights into the assembly of the heeromeric 

pyridoxalphosphate synthase from Plasmodium falciparum, J. Mol. Biol., 2007, 

374:732-748. 

11) Stueckler, C., Hall, M., Ehammer, H., Pointner, E., Kroutil, W., Macheroux, P., 

and Faber, K.: 

Stereo-complementary bioreduction of α,β-unsaturated dicarboxylic acids and 

dimethly esters using enoate reductases: Enzyme- and substrate-based 

stereocontrol, Org, Lett., 2007, 9:5409-5411. 

Awards 

Gernot Rauch: Best paper award 2006/07 for doctoral students of the key research area 

“Life Science Technology” of the Graz University of Technolgy (July 2007) 

Sonja Sollner: Winner of the 1 st prize for best presentation at the annual graduate 

seminar of the PhD programme “Molecular Enzymology” (May 2007) 

14

Cell Biology Group 

Group leader: Günther Daum 

Graduate students: Sabine Rosenberger, Andrea Wagner, Tamara Wriessnegger, 

Irmgard Schuiki, Tibor Czabany, Melanie Connerth, Sona Rajakumari, 

Karlheinz Grillitsch, Miroslava Spanova 

Diploma student: Susanne Horvath 

Technician: Claudia Hrastnik 

Visiting scientists: Miroslava Spanova (Bratislava, Slovak Republic; Erasmus 

fellowship), Gordana Canadi Juresic (Rijeka, Croatia; CEEPUS fellowship) 


A fundamental problem of cell biology is the biogenesis of subcellular 

structures. Along that line, studies in our laboratory are focused on the synthesis of 

lipids and their assembly into organelle membranes of the yeast Saccharomyces 

cerevisiae. During the last decades, baker’s yeast has proven as a valuable and highly 

reliable model for eukaryotic cells. We make use of the advantages of this experimental 

system to combine biochemical, molecular and cell biological methods addressing 

problems of lipid metabolism and biomembrane formation. 

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, (ii) neutral lipid storage in 

lipid particles and mobilization, and (iii) characterization of organelle membranes from 

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 and (iv) the CDP-ethanolamine pathway (Kennedy pathway) in the 

endoplasmic reticulum. 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 

15

performed. Three components identified through these screenings were recently 

characterized in more detail, namely Oxa1p, Mrpl22p and Vps4p. Oxa1p is a 

component of the inner mitochondrial membrane and involved in the assembly of 

proteins into this compartment (Figure 1). Deletion of OXA1 causes a defect in the 

import of Psd1p, PtdSer decarboxylase 1, into mitochondria. Since Psd1p is the major 

enzyme of PtdEtn formation in the yeast, the oxa1 mutation results in a marked 

decrease of PtdEtn compared to wild type. 

Psd1p 

Targeting IM Sorting β-Subunit LGST -Subunit 

++++ 

+ 

TOM 

OMM 

TIM22 

TIM23 

Oxa1p 

Mba1p 

Psd1p 

IMM 

Psd1p 

Figure 1: The Oxa1p route of Psd1p import into mitochondria 

Mrpl22p is a component of mitochondrial ribosomes. Deletion of the respective 

gene causes defects comparable to psd1, namely decrease of cellular and mitochondrial 

PtdEtn and defects in mitochondrial respiration. The molecular reason for these 

findings is not yet known. Vps4p is a component of the machinery governing protein 

traffic to yeast vacuoles. In strains deleted of VPS4 an alternative pathway of protein 

transport to vacuoles, the so-called autophagy/cytosol to vacuole targeting (Cvt) 

pathway, is induced. The latter route requires PtdEtn binding to Atg8p, a component 

involved in autophagy. Thus, shortage of PtdEtn in respective mutants compromises 

the autophagic process and causes a growth defect. 

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 themselves. We employed 

yeast mutants bearing defects in the three pathways of PtdEtn synthesis, which are 

distributed among mitochondria, endoplasmic reticulum and Golgi. These experiments 

16

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

be studied in future investigations. 

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. 

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

and Lro1p contribute to the formation of lipid depots (Figure 2). We have to distinguish 

between de novo formation of lipid particles on one hand and incorporation of neutral 

lipids into preformed lipid particles on the other hand. Using a dga1 lro1 are1 are2 

quadruple mutant and the related set of triple mutants we performed cell biological and 

Figure 2: Pathways of triacylglycerol (TAG) and steryl ester (SE) metabolism in the 

yeast. DAG: diacylglycerol; FFA: free fatty acids; PL: phospholipid. 

17

iochemical experiments to address questions related to new formation and growth of 

lipid particles. Depending on the mutant used we were able to obtain lipid particles of 

different composition. These different types of lipid particles were investigated using 

biochemical, cell biological and biophysical methods. 

Mutants described above 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 3). 

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 3: 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. 

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. 

Our investigations demonstrated that each SE hydrolase exhibits some 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 

18

iosynthesis 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 C22:0 and C26:0 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 turnover 

compared to wild type. Competition experiments with labeled palmitic acid (C16:0) and 

non-radiolabeled cerotic acid (C26:0) demonstrated that the C26:0 fatty acid competed 

with C16:0 fatty acid labeling of sphingolipids. Thus, we hypothesize that stored TAG 

can be a fatty acid donor for sphingolipid formation and TAG lipases contribute to this 

pathway of the yeast. 

Another aspect addressed in our laboratory is characterization of yeast TAG 

lipases with special emphasis on the topology of these proteins on the surface of lipid 

particles where these proteins occur. This study is paralleled by topology studies of 

other proteins located to the lipid particle surface, especially Erg1p, a squalene 

epoxidase, which has been studied in our laboratory before. Preliminary studies 

addressing accumulation of squalene in the yeast under certain conditions revealed that 

this component is localized to different subcellular sites depending on the cell biological 

and metabolic status of the yeast. 

Characterization of organelles from the yeast Pichia pastoris 

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 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 from Pichia pastoris grown on different carbon sources (Figure 4). The 

major aims of this project were (1) qualitative and quantitative analysis of 

phospholipids, fatty acids and sterols from mitochondrial membranes; and (2) analysis 

of mitochondrial subfractions from Pichia pastoris grown on different carbon sources. 

More recently, experiments were started to study selected lipid synthesizing enzymes 

from Pichia pastoris with emphasis on the phosphatidylethanolamine biosynthetic 

machinery. Preliminary data are a first step towards the understanding of the Pichia 

pastoris lipidome which will provide initial insight into the variety of membrane 

components of this yeast. 

19

A 

B 

V 

N 

M 

Figure 4: Electron microscopy of Pichia pastoris X33 wild type grown on glycerol (A). 

Under these conditions mitochondria are well expressed. B: isolated mitochondria from 

Pichia pastoris grown on glycerol. M, mitochondria; V, vacuole; N, nucleus; Bars. 0.5 

and 2 µm. Pictures shown be curtsey of E. Ingolic, FELMI, TU Graz, Austria 

Doctoral Thesis completed 

Sabine Rosenberger 

Phosphatidylethanolamine (PtdEtn) is an essential component in the yeast 

Saccharomyces cerevisiae required for function and integrity of mitochondrial 

membranes, for protein import/assembly into the inner mitochondrial membrane, for 

GPI-anchor synthesis and vacuolar targeting. Biosynthesis of PtdEtn can be 

accomplished by three different pathways, with the major route mediated by 

phosphatidylserine decarboxylase 1 (Psd1p) residing in the inner mitochondrial 

membrane (IMM). Deletion of PSD1 leads to a significant decrease of cellular and 

mitochondrial PtdEtn thereby conferring a petite phenotype, which is characterized by 

the loss of respiratory capacity and mitochondrial DNA. To identify a link between the 

petite phenotype and disturbed lipid homeostasis in mitochondria a petite yeast strain 

collection was screened for mutants with abnormal phospholipid patterns. This 

approach unveiled some candidate genes, among them MRPL22. Deletion of MRPL22 

leads to a decreased amount of cellular and mitochondrial PtdEtn similar to a psd1 

deletion strain. Another major part of this thesis was focused on the assembly of PtdEtn 

into peroxisomal membranes. To define the traffic routes of PtdEtn from their site of 

synthesis to peroxisomes single and multiple mutants bearing defects in the different 

pathways of PtdEtn synthesis were used. PtdEtn formed through all three pathways in 

different subcellular membranes can be supplied to peroxisomes with comparable 

efficiency. 

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 

V. Tomekova, Department of Medical Chemistry, Biochemistry and Clinical 

Biochemistry, Faculty of Medicine UPJS, Košice, Slovak Republic 

20

T. Hoefken, Institute of Biochemistry, University of Kiel, Germany 

D. Warnecke, Institute of Botany, University of Hamburg, Germany 

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 

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 

Phosphatidylethanolamine, a key lipid of the yeast 

Comenius University, Bratislava, Slovak Republic, 19 th April 2007 

2) G. Daum 

Neutral lipid storage and mobilization in the yeast 

Technical University, Bratislava, Slovak Republic, 20 th April 2007 

3) G. Daum, T. Czabany, A. Wagner, M. Spanova, S. Rajakumari, K. Grillitsch and 

K. Athenstaedt 

Playing the yeast lipid game 

8 th Yeast Lipid Conference, Torino, Italy, 10-12 May 2007 

4) G. Daum, K. Athenstaedt, A. Wagner, T. Czabany, K. Grillitsch, S. Rajakumari, 

M. Spanova, D. Zweytick and E. Ingolic 

Formation and mobilization of neutral lipid depots in the yeast 

3 rd European Federation of Biotechnology Conference on Physiology of Yeast and 

Filamentous Fungi, Helsinki, Finland, 13-16 June 2007 

5) G. Daum 

The Life Cycle of Neutral Lipids: Synthesis, Storage and Degradation 

Gordon Research Conference “Molecular & Cellular Biology of Lipids“, 

Waterville Valley Resort, NH, USA, 22-27 July 

Publications 

1) Daum, G., Wagner, A., Czabany, T. and Athenstaedt, K. 

Dynamics of neutral lipid storage and mobilization in the yeast 

Biochimie 89 (2007) 243-248 

21

2) Sollner, S.; Nebauer, R.; Ehammer, H.; Prem, A.; Deller, S.; Palfey, B. A.; Daum, 

G. and Macheroux, P. 

Lot6p from Saccharomyces cerevisiae is a FMN-dependent reductase with a 

potential role in quinone detoxification 

FEBS J. 274 (2007) 1328-1339 

3) Czabany, T., Athenstaedt, K. and Daum, G. 

Synthesis, storage and degradation of neutral lipids in yeast 

Biochim. Biophys. Acta 1771 (2007) 299-309 

4) Wriessnegger, T, Gübitz, G, Leitner, E, Ingolic, E, Cregg, J, de la Cruz, B. J. and 

Daum, G. 

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on 

different carbon sources 


5) Perktold, A., Zechmann, B., Daum, G. and Zellnig, G. 

Organelle association visualized by three-dimensional ultrastructural imaging of 

the yeast cell 

FEMS Yeast Research 7(2007) 629-638 

6) Nebauer, R., Rosenberger, S. and Daum, G. 

Phosphatidylethanolamine, a limiting factor of autophagy in yeast strains bearing a 

defect in the carboxypeptidase y pathway of vacuolar targeting 

J. Biol. Chem. 282 (2007) 16736-16743 

7) Nebauer, R, Schuiki, I., Kulterer, B., Trajanoski, Z. and Daum, G. 

The phosphatidylethanolamine level of yeast mitochondria is affected by the 

mitochondrial components Oxa1p and Yme1p 

FEBS J. 274 (2007) 6180-6190 

8) Daum, G, Wagner, A., Czabany, T, Grillitsch, K. and Athenstaedt, K. 

Lipid storage and mobilization pathways in yeast 

Novartis Foundation Symposium No.286 (2007), 142-154 

Award 

Ruth Nebauer, a former PhD student, received the Best Paper Award 2006/07 for 

doctoral students of the Key Research Area “Life Science Technology” of the Graz 

University of Technology (July 2007) 

22

Molecular Biology Group 

Karin Athenstaedt 

Lipid particles – more than simple storage compartments 

(Professorial dissertation) 

Lipid particles are intracellular compartments consisting of a neutral lipid core 

surrounded by a phospholipid monolayer with few proteins embedded. In former days, 

the only function ascribed to these particles was that of a simple storage compartment 

containing backup molecules, mainly triacylglycerols and steryl esters, which are 

mobilized when nutrients are no longer provided by the environment. This view, 

however, had to be revised, since investigations during the last decade demonstrated 

active participation of lipid particle proteins in lipid metabolism. My work deals with 

identification and characterization of several lipid particle proteins of the yeast 

Saccharomyces cerevisiae which are involved in processes such as phosphatidic acid 

biosynthesis, triacylglycerol and sterol formation, and triacylglycerol mobilization. 

Analysis of the lipid particle proteome of the oleaginous yeast Yarrowia lipolytica 

revealed that most proteins are counterparts of lipid particle polypeptides of 

Saccharomyces cerevisiae. Comparison of the yeasts’ lipid particle proteome with that 

of higher eukaryotic cells indicates that participation in lipid metabolism is a general 

feature of this compartment. 

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 

Figure 1: “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). 

23

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. 

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 yeast Saccharomyces cerevisiae encoded by the 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 must 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 this 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 mutants were analyzed by fluorescence 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. 

This work was performed at OHSU (Oregon Health and Sciences University, 

Portland, Oregon) during my post doctoral studies in 2007. 

Lipid metabolism of the yeast Yarrowia lipolytica 

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to produce 

and store triacylglycerols. For this reason, this microorganism has become attractive for 

industrial applications such as production of single cell oil. In a former project, we 

investigated lipid particles of the oleaginous yeast with respect to their lipid and protein 

composition. Analysis of the lipid particle proteome identified major lipid particle 

24

proteins. The “standard” proteome of lipid particles isolated from glucose grown cells 

was complemented by several others when cells were grown on oleic acid. Homology 

searches using the ORFs coding for lipid particle polypeptides of Yarrowia lipolytica as 

a query revealed that most lipid particle proteins are counterparts of proteins involved in 

lipid metabolism in Saccharomyces cerevisiae. However, since the function of these 

proteins was only deduced upon homology, we are currently investigating the “true” 

function of these proteins with a special emphasis on those involved in triacylglycerol 

synthesis. 

Figure 2: 

Accumulation of neutral 

lipids in lipid particles of 

Yarrowia lipolytica wildtype 

cell either grown on 

oleic acid (left) or glucose 

(right). Neutral lipids are 

stained with the fluorescent 

lipophilic dye NileRed®. 

Special Activities 

Habilitation for the subject Biochemistry 

Postdoctoral studies at the Oregon Health and Sciences University (OHSU), Portland, 

Oregon, USA 

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 

J. M. Thevelein, Centre for Food and Microbial Technology, Department of Microbial 

and Molecular Systems, Katholeke Universiteit Leuven, Heverlee, Belgium 

Publications 

1) Daum, G., Wagner, A., Czabany, T. and Athenstaedt, K. 

Dynamics of neutral lipid storage and mobilization in the yeast 

Biochimie 89 (2007) 243-248 

2) Czabany, T., Athenstaedt, K. and Daum, G. 

Synthesis, storage and degradation of neutral lipids in yeast 


3) Daum, G, Wagner, A., Czabany, T, Grillitsch, K. and Athenstaedt, K. 

Lipid storage and mobilization pathways in yeast 

Novartis Foundation Symposium No.286 (2007), 142-154 

25

Biophysical chemistry group 

Group leader: Albin Hermetter 

Post-docs: Heidrun Susani-Etzerodt, Jo-Anne Rasmussen 

Graduate students: Maria Morak, Jessica Schatte, Maximilian Schicher, Ute Stemmer 

Undergraduate students: Peter Krempl, Andreas Gasser, 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 

26

specific action of sphingolipid 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 biologically 

active compounds are very likely to interfere 

directly with various signaling components 

inside the cells, which finally decide about cell 

growth or death. Investigations will be 

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 can lead to serious 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. 

27

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 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 the 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 

28

in gel-digest, the isolated peptides were analyzed by nanoHPLC-MS/MS. So far, the 

entire lipolytic proteome of mouse liver has also been determined. The function of 

overexpressed enzyme candidates is currently being identified using defined lipid 

substrates 

2.2. Protein microarrays – Enzyme chips 

In the framework of the Kplus Research Centre 

Applied Biocatalysis (TU Graz), we develop 

"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 affinity 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 of the 

Angewandte 

Biokatalyse 

Kompetenzzentrum GmbH 

bio chips to the investigation of real analytical problems. These systems will be used for the 

screening of lipases with specific substrate acceptance. 

International Cooperations: 

T. Hianik, Department of Biophysics and Chemical Physics, Comenius University, 

Bratislava, Slovakia 

P. Deigner, SIRS Lab, Jena, Germany 

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 

Diploma thesis completed: 

Peter Krempl: Development of an electrophoresis method for differential analysis of 

lipolytic enzymes 

In functional proteomics, "activity-based probes" (ABPs) are essential tools for labelling 

and detection of biocatalysts. Typical ABPs contain a specific binding group, a reactive 

functional moiety and a reporter group (e.g. a fluorescent dye) for protein detection. 

This study describes the chemical synthesis and application of a set of matched ABPs 

for detection of lipases and esterases, that can be used for a precise comparative 

29

characterisation of several lipolytic enzymes in a complex proteome. For this purpose, 

proteome samples are exposed to the ABPs, followed by electrophoretic protein 

separation, acquisition and software-assisted analysis of the fluorescence images of the 

obtained protein patterns. The new method was validated using defined mixtures of 

purified lipase and esterase preparations as well as natural tissue samples. The 

statistically valid thresholds for the detection of differences between the compared 

samples were determined. 

Research Projects: 

TIG Research center Applied Biocatalysis (coordinated by TU Graz): Novel lipases with 

specific substrate acceptance. 

bm:bwk (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 Doctoral Program Molecular Enzymology: Functional enzyme analysis 

(coordinated by KFU Graz). 

FWF Special Research Program (SFB) LIPOTOX (coordinated by KFU Graz): Toxicity 

of oxidized phospholipids in macrophages. 

Invited lectures: 

1) Effects of oxidized phospholipids on vascular smooth muscle cells. University of 

Linz, Austria, March 6, 2007 

2) Novel fluorescent probes for lipids and lipases. 10 th International Conference on 

Methods and Applications of Fluorescence Spectroscopy, Imaging and Probes 

(MAF-10), Salzburg, Austria, 9 – 12 September 2007. 

3) Natürliche Antioxidantien in Lebensmitteln. Salzburger Gesundheitstage, 

Salzburg Austria, April 21, 2007. 

4) Apoptotic signaling of oxidized phospholipids in vascular smooth muscle cells 

and macrophages. Hermann Esterbauer Meeting, Graz, Austria, October 15, 2007 

5) Apoptotic effects of oxidized phospholipids on vascular smooth muscle cells. 

COST Meeting Bioradicals, Rome, Italy, September 14, 2007 

Publications: 

1) Moumtzi A, Trenker M, Flicker KH, Zenzmaier E, Saf R and Hermetter A 

Import and fate of fluorescent analogs of oxidized phospholipids in vascular 

smooth muscle cells 

J.Lipid Res. 48: 565 – 582, 2007 

2) Yates RM, Hermetter A, Taylor GA and Russell DG 

Macrophage activation down-regulates the degradative capacity of the phagosome 

Traffic 8: 241 – 250, 2007 

30

3) Wehinger A, Tancevski I, Schgoer W, Eller P, Hochegger K, Morak M, Hermetter 

A, Ritsch A, Patsch JR and Foeger B 

Phospholipid transfer protein augments apoptosis in THP-1-derived macrophages 

induced by lipolyzed hypertriglyceridemic plasma 

Arterioscler.Thromb.Vasc.biol. 27: 908 – 915, 2007 

4) Werner ER, Hermetter A, Prast H, Golderer G and Werner-Felmayer G 

Widespread occurrence of glyceryl ether monooxygenase activity in rat tissues 

detected by a novel assay 

J.Lipid Res. 48: 1422 – 1427, 2007 

5) Tilz GP, Wiltgen M, Demel U, Faschinger C, Schmidinger H and Hermetter A 

Insights into molecular medicine: Development of new diagnostic and prognostic 

parameters 

Wien.Med.Wochenschr. 157: 122 – 129, 2007 

6) Fruhwirth GO, Loidl A and Hermetter A 

Oxidized phospholipids: From molecular properties to disease 

Biochim.Biophys.Acta 1772: 718 – 736, 2007 

7) Birner-Gruenberger R and Hermetter A 

Activity-based proteomics of lipolytic enzymes 

Curr.DrugDiscov.Technol. 4: 1 – 11, 2007 

8) Fruhwirth GO and Hermetter A 

Seeds and oil of the Styrian oil pumpkin: Components and biological activities 

Eur.J.LipidSci.Technol. 109: 1128 – 1140, 2007 

31

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 und Molekularer Biomedizin 



Molekulare Physiologie 2 VO Macheroux P 

Projektlabor Bioanalytik 6 PR Daum G, Hermetter 


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 



Anleitung zu wissenschaftlichen Arbeiten aus 

Biochemie und Molekularer Biomedizin 



Spezielle Kapitel der Biochemie 1 VO Daum G, Hermetter 


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 


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 

32

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

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