Graz University of Technology Austria Institute of Biochemistry ...

Graz University of Technology 

Austria 

Institute of Biochemistry 

Annual Report 2010 

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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 

Michael Murkovic (Associate Professor) 

michael.murkovic@tugraz.at; Tel.: +43-(0)316-873-6495 

Karin Athenstaedt (Independent Group Leader) 

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

Assistants 

Ines Waldner-Scott 

ines.waldner-scott@tugraz.at; Tel.: +43-(0)316-873-6454 

Tanja Knaus 

tanja.knaus@tugraz.at; Tel.: +43-(0)316-873-6463 

Alexandra Binter 

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

Silvia Wallner 

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 

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 

Alma Ljubijankic; alma.ljubijankic@tugraz.at; Tel.: +43-(0)316-873-6460 or 6498 

Eva Maria Pointner; eva.pointner@tugraz.at; Tel.: +43-(0)316-873-6453 

Leo Hofer (Workshop); leo.hofer@tugraz.at; 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 biosciences were 

then concentrated. 

1945 G. 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 Prof. F. PREGL and Prof. F. 

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 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 at the Graz 

University of Technology, 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 

independent institute. 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 headed by Prof. LAFFERTY. 

1973 Dr. F. PALTAUF, docent at the Karl-Franzens-University Graz, was appointed 

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. G. DAUM, S. D. KOHLWEIN, and A. HERMETTER 

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joined the institute. All three young scientists were given the chance to work as post 

docs in renown 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 technology were held by C. WEBER and H. SALOMON. 

Hence the institute was renamed Institute of Biochemistry and Food Chemistry. 

1990 The institute moved to a new building at Petersgasse 12. The move was accompanied 

by the expansion of individual research groups and the acquisition of new equipment 

essential for the participation in novel collaborative efforts at the national and 

international level. 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 into the Institute of 

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

Technology (Head Prof. PFANNHAUSER). 

2001 After F. PALTAUF’s retirement, in September 2001, G. DAUM was elected head of 

the institute. S. D. KOHLWEIN was appointed full professor of biochemistry at the 

Karl-Franzens University 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 protein biochemistry and enzymology and shall strengthen the 

already existing activities in this area. 

2007 K. ATHENSTAEDT, a long-time associate of Prof. DAUM, received the venia 

legendi for biochemistry. Dr. Athenstaedt was the first woman to complete the 

traditional habilitation at the Institute of Biochemistry. 

2009 After the retirement of Prof. PFANNHAUSER in 2008, the Institute of Food 

Chemistry and Technology was disbanded and the research group of Prof. M. 

MURKOVIC joined the Institute of Biochemistry increasing the number of 

independent research groups to five. 

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Highlights of 2010 

In 2010, Peter Macheroux took a break from his teaching duties and spent most of the year 

from mid-February until the end of July on sabbatical leave in the Dept. of Chemistry & 

Chemical Biology at Cornell University (hosted by Prof. Steve Ealick). This was a truly 

unique experience not just because of the opportunity to acquire insight into new scientific 

methods but also in terms of getting to know new colleagues and customs, such as 

commencement day where students obtain their academic degree during a traditional 

celebration. In spring 2010 the Austrian Science Fund (FWF) approved a new research project 

to investigate the interaction of quinone reductase with the proteasome that controls the 

localization of some transcription factors (Project P22361: “Mechanism of redox controlled 

protein degradation”). Finally, Dr. Andreas Winkler, a former PhD student of the PhD 

program “Molecular Enzymology” and currently a postdoctoral fellow at the Max-Planck 

Institute of Molecular Medicine in Heidelberg received the dissertation award 2010 of the 

Austrian Society for Molecular Biosciences and Biotechnology (ÖGMBT) for the thesis he 

has completed in our institute the year before. 

Impressions from the sabbatical of P. Macheroux 

Commencement day at Cornell in May 2010 

Karlheinz Grillitsch was the 100 th student to receive 

his PhD at the Institute of Biochemistry. Opponents 

were K. Lohner (left) and G. Daum (right of the 

candidate); chairman A. Hermetter (right). 

In the group of Günther Daum, three new projects were started in 2010. Whereas the FWF 

project P23029 is devoted to the fundamental investigation of yeast lipases, the Translational 

Research Project TRP009 addresses applied aspects of Pichia lipidomics. Finally, the lab of 

Günther Daum is involved in investigations of protein expression in the framework of the 

Austrian Centre of Industrial Biotechnology (ACIB). A highlight in graduate student 

education was the completion of the Doctoral Thesis of Karlheinz Grillitsch because this was 

the 100 th PhD degree received at the Institute of Biochemistry, TU Graz. In 2010, Günther 

Daum started his three year term as President of the International Conference on the 

Bioscience of Lipids (ICBL). In May 2010, he was Organizer and Chairman of the FEBS 

Workshop Microbial Lipids: From Genomics to Lipidomics, in Vienna, Austria. This was the 

first conference of this type, but due to its success with more than 100 participants the next 

meeting has already be planned for 2012 in Bern, Switzerland. 

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In 2010 Karin Athenstaedt continued her work in our institute as an independent 

researcher. With her project she set the cornerstone to establish her own research group. 

Karin Athenstaedt’s work is devoted to lipid metabolism in yeast. 

Basic research in Albin Hermetter’s group was performed in three projects focusing on lipid 

(patho)physiology. His contribution to the GOLD project (speaker R. Zechner, ZMB, 

University of Graz, funded by the GEN-AU program of the BM.W_F) is devoted to the 

functional proteomic analysis of (phospho)lipases relevant to lipid-associated disorders. In the 

SFB LIPOTOX (FWF project, speaker R. Zechner) and the EuroMEMBRANE consortium 

OXPHOS (speaker P. Kinnunen, Aalto University, Helsinki), A. Hermetter studies the toxicity 

of oxidized phospholipids in the cells of the arterial wall (funded by FWF). Research in this 

field is also supported by the PhD program “Molecular Enzymology” (FWF). Ute Stemmer 

who is a PhD student in the SFB LIPOTOX was awarded the prize for the best oral 

presentation by young researchers entitled “Oxidized phospholipids: uptake and targeting in 

RAW 264.7 macrophages” at the International Conference on the Bioscience of Lipids in 

Bilbao, Spain, September 7 – 11, 2010. 

K. Athenstaedt 

M. Murkovic 

Ute Stemmer (in the center) received the BBA Award presented by W. 

Dowhan (left; BBA Executive Editor) for the best oral presentation by young 

researchers at the International Conference on the Bioscience of Lipids in 

Bilbao, Spain, September 7 – 11, 2010. 

In 2010, Alan Zeb finished his PhD Thesis in the group “Chemistry of Functional Foods” 

directed by Michael Murkovic in which the interaction of carotenoids with triglycerides from 

edible oils was characterized in detail. The pro- and antioxidant action of carotenoids was 

evaluated by a detailed analysis of the oxidation products by LC-MS. Several oxidation 

products of triglycerides were newly identified. Four manuscripts were published on this 

topic. In 2010 the biannual conference of the Austrian Food Chemists was held at Schloss 

Seggau near Leibnitz. At this conference around 100 participants from Austria and the 

neighboring countries discussed the issue of food quality on the molecular level. 

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Biochemistry group 

Group leader: Peter Macheroux 

Secretary: Annemarie Portschy 

Postdoctoral Fellow: Ines Waldner-Scott 

PhD students: Thomas Bergner, Alexandra Binter, Venugopal Gudipati, Tanja Knaus, Wolf- 

Dieter Lienhart, Silvia Wallner 

Technicians: Eva Maria Pointner, Steve Stipsits, Rosemarie Trenker-El-Toukhy 

Alumni 2010: Katrin Fantur (PhD) 

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 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 also 

frequently use MALDI-TOF and ESI mass spectrometry, protein purification techniques 

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

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

Berberine bridge enzyme & other flavin-dependent plant oxidases 

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: 

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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. 

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). 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). Currently, Silvia 

Wallner investigates alternative covalent modifications in the 8α-position in BBE variants 

that contain either aspartate or tyrosine instead of a histidine. In collaboration with Prof. Toni 

Kutchan at the Donald Danforth Plant Science Center in St. Louis, we have identified other 

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plant genes that apparently encode flavin-dependent oxidases. These genes are currently 

expressed in our laboratory in order to characterize the role of the enzymes in alkaloid 

biosynthesis (thesis project of Silvia Wallner). 

Dipeptidylpeptidase III 

Dipeptidyl-peptidases III (DPPIII; EC 3.4.14.4) are Zn-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. Previously, 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 yeast 

protein. This work revealed that yeast DPPIII features a novel protein topology. 

Structure of human DPPIII in its open form (right) and peptide liganded (closed) form 

In collaboration with a structural genomics group in Toronto led by Dr. Sirano Dhe-Paganon, 

Gustavo Arruda has solved the structure of the human enzyme both in its open (right, above) 

and closed conformation (left, above). The latter structure was obtained by co-crystallization 

of a tightly binding peptide to an inactive variant of human DPPIII. These two new structures 

constitute a major breakthrough in our effort to understand the physiological role of the 

enzyme and pave the way for the development of potentially useful inhibitors of the enzyme 

(Gustavo Arruda’s thesis project in Prof. Gruber’s laboratory supported by Alexandra Binter). 

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Luciferase and LuxF 

The emission of light by biological species (bioluminescence) is a fascinating process found 

in diverse organisms such as bacteria, funghi, insects, fish, limpets and nematodes. In all cases 

the bioluminescent process is based on a chemiluminescent reaction in which the chemical 

energy is (partially) transformed into light energy ("cold light"). All bioluminescent processes 

require a luciferase, i.e. an enzyme catalyzing the chemiluminescent reaction, and a luciferin, 

which can be considered a coenzyme. During the bioluminescent reaction the luciferin is 

generated in an excited state and serves as the emitter of light energy. In our laboratory, we 

are interested in the bioluminescence of marine photobacteria. In these bacteria, luciferase is 

composed of an alpha/beta-heterodimeric protein, which binds reduced flavinmononucleotide 

(FMN) as the luciferin. The reduced FMN reacts with molecular dioxygen to a hydroperoxide 

intermediate with subsequent oxidation of a long-chain fatty aldehyde (e.g. tetradecanal) to 

the corresponding fatty acid (e.g. myristic acid). During this oxidation process, an excited 

flavin intermediate is generated which emits light. Some marine photobacteria possess an 

additional protein called LuxF which was found in complex with a myristylated flavin 

derivative where the C-3 atom of myristic acid is covalently attached to the 6-position of the 

flavin ring system. It was postulated that this flavin adduct is generated in the luciferase 

catalyzed bioluminescent reaction. Furthermore, it was speculated that LuxF sequesters the 

myristylated flavin adduct in order to prevent inhibition of the bioluminescent reaction. 

However, both hypotheses have not been tested on a biochemical or physiological level yet. 

Hence, in this study we will design and perform experiments to examine the putative 

generation of myristylated FMN through the luciferase reaction (thesis project of Thomas 

Bergner supported by Steve Stipsits) 

Structure of a LuxF dimer in the absence (red) and presence (blue) of the myristylated flavin 

derivative (pdb code 1NFP) 

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, organ transplant recipients and 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 

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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 synthesis of 

aminohexuronic acid 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 at 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. Co-crystallization of NikO with fosfomycin yielded rod – like crystals diffracting 

up to 2.5 Å. A synchrotron dataset was measured at the Swiss Light Source and the structure 

was solved by molecular replacement using UDP-N-acetylglucosamine enolpyruvyl 

transferase (PDB code: 2rl1) as a model. Two molecules were found in the asymmetric unit 

exhibiting an inverse α,β-barrel fold with helices forming the tightly packed core and sheets 

shielding the hydrophobic core from the solvent: 

Eech chain is comprised of two inverse α,β-barrel subunits, which are connected by a hinge 

region. The final structure was refined to final R/R free values of 17% and 19%, respectively. 

Crystallization trials of NikO in the presence of its product, 3’-EP-UMP, are currently under 

way. 

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The genes that are co-transcribed with nikO have been reported and are designated as 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 are cloned, expressed and the 

proteins purified for biochemical characterization and crystallization (thesis project of 

Alexandra Binter and Gustav Oberdorfer in Prof. Gruber’s laboratory). 

Lot6p – a redox regulated switch of the proteasome 

During our previous studies of bacterial quinone reductases, we have also investigated the 

biochemical properties of the yeast homolog Lot6p. Despite the availability of a threedimensional 

structure for Lot6p (1T0I), the physiological role of the enzyme was unclear. 

Our recent studies have now demonstrated that the enzyme rapidly reduces quinones at the 

expense of a reduced nicotinamide cofactor, either NADH or NADPH. In order to further 

characterize the cellular role of Lot6p, we have carried out pull-down assays and identified 

the 20S core particle of the yeast proteasome as 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 (see scheme below). 

reduced 

Yap4p 

oxidized 

Yap4p 

Yap4p 

Lot6p 

α 

β 

β 

α 

Oxidation by 

e.g. quinones 

Very slow with oxygen O 2 

oxygen 

Lot6p 

α 

β 

β 

α 

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 (postdoctoral project of Ines 

Waldner-Scott; thesis project of Wolf-Dieter Lienhart and Venugopal Gudipati). 

Zn-dependent Alkylsulfatases 

Hydrolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize 

sulfur. Three classes of sulfatases - divided according to their reaction mechanism - have been 

discovered, and the recently elucidated structure of SdsA1 from Pseudomonas aeruginosa is 

another example of the widely occurring family of metallo-ß-lactamases. This group of 

alkylsulfatases is characterized by two Zn 2+ atoms in the active site which activate a water 

molecule for nucleophilic attack on the sulfate group. SdsA1 mainly cleaves long-chain 

primary alkylsulfates (preferred substrate is dodecylsulfate) by stereoinversion. In other 

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words, the hydroxyl group attacks the carbon atom in the course of the reaction. Recently, a 

novel enzyme could be identified in Pseudomonas DSM 6611 (termed PISA1 = Pseudomonas 

inverting alkylsulfatase 1) which mainly cleaves secondary alkylsulfates, for example 2- 

octylsulfate exhibiting stereopreference for the (R)-stereoisomer. In contrast to the majority of 

hydrolases, which do not alter the stereochemistry of the substrate during catalysis, PISA1 is 

an attractive enzyme for the deracemisation of sec-alcohols. 

Analysis of the crystal structures of PISA1 and SdsA1 showed that the overall structure of 

both proteins is virtually identical and both enzymes largely share the same active-site 

architecture, such as a sulfate binding site (composed of two Arg), a nucleophile site 

composed of a binuclear Zn 2+ -cluster typical for metallo-ß-lactamases and an Asn/Thrhydrogen 

binding network for substrate positioning. However, the active site of PISA1 

features several conspicuous amino acid exchanges (see figure below: in blue active side 

residues in SdsA1 and green those in PISA1). 

Phe/Gly 

Tyr/His 

Met/Ser 

Met/Ser 

Leu/Pro 

Ala/Ile 

Tyr/Ser 

Tyr/Ser 

Ala/Ile 

These amino acids are now subject of an extensive mutagenesis program to define their role in 

governing substrate preference of the reaction. This project is a close collaboration with Profs. 

Faber (biocatalysis) and Wagner (structure determination) from the University of Graz (thesis 

project of Tanja Knaus in our laboratory and Markus Schober in Prof. Faber’s laboratory). 

Doctoral thesis completed 

Katrin Fantur: Friend or Foe: Iminosugars as inhibitors and pharmacological chaperones of 

the human lysosomal acid β-galactosidase 

G M1 -gangliosidosis (GM1) and Morquio B disease (MBD) are rare, hereditary lysosomal 

storage disorders caused by mutations in the gene GLB1. Its main gene product, human 

lysosomal acid β-galactosidase (β-Gal) degrades N-linked oligosaccharides present in 

glycoproteins, GM1-gangliosides in the brain, and keratan sulfate in connective tissues. While 

GM1 is a phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone 

disease without effects on the central nervous system. Some mutations in the GLB1 gene 

produce stable β-Gal precursors, normally transported and processed to mature, 

intralysosomal β-Gal, while others affect precursor stability and intracellular transport 

resulting in premature protein degradation. Several misfolded enzymes were shown to be 

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sensitive to stabilization by iminosugars, which bind at the active site to provide the proper 

conformation. Thus the stabilized protein may escape from degradation processes, and reach 

the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). 

In this work the influence of novel derivatives of 1-deoxygalactonojirimycin (DGJ) 

on the β-Gal activity of cultured GM1 and MBD skin fibroblasts was examined. Furthermore, 

the effect of selected compounds on natural substrate degradation in GM1 and MBD cells was 

determined. Several novel iminosugars acting as pharmacological chaperones of β-Gal in 

specific GM1 fibroblasts were discovered and described in this work. One specific compound, 

DLHex-DGJ, proved to be a potent competitive inhibitor of β-Gal in vitro, and this work 

describes its effects on activity, protein expression, maturation and intracellular transport in 

vivo in 13 fibroblast lines with GLB1 mutations. DLHex-DGJ significantly increased the 

catalytic activity in six GM1 cell lines, and normalization of transport and lysosomal 

processing of β-Gal precursors was demonstrated for selected cell lines. Furthermore, 

DLHex-DGJ and another, similar compound successfully reduced the level of internalized 

radiolabeled G M1 -gangliosides in a specific GM1 cell line, suggesting that reduction of stored 

material is possible under certain conditions. 

Specific antibodies, directed against human β-Gal, were developed with the aid of 

previously published protocols, and novel approaches to obtain large amounts of the purified 

human enzyme were tested. Two novel polyclonal anti-β-Gal peptide antibodies were 

produced and expression of human β-Gal in E. coli cells may provide the basis for further 

development of antibodies directed against the human enzyme. Large parts of this thesis were 

carried out at the University hospital under the guidance and supervision of Prof. Pascke. 

International cooperations 

Maria Abramic, Ruder Boskovic Institute Zagreb, Croatia 

Steve Ealick, Cornell University, Ithaca, U.S.A. 

Toni Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A. 

Shwu Liaw, National Yang-Ming University, Taipei, Taiwan 

Matthias Mack, Hochschule Mannheim, Germany 

Bruce Palfey, University of Michigan, Ann Arbor, U.S.A. 

Research projects 

FWF P22361: “Mechanism of redox controlled protein degradation” 

FWF P19858: “Enzymes of nikkomycin biosynthesis” 

FWF-Doktoratskolleg “Molecular Enzymology” 

WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49 

family” 

Publications 

1) Durchschein, K., Ferreira-da Silva, B., Wallner, S., Macheroux, P., Kroutil, W., Glueck, 

S. M., Faber, K.: The flavoprotein-catalyzed reduction of aliphatic nitro-compounds 

represents a biocatalytic equivalent to the Nef-reaction, Green Chemistry, 2010, 12:616- 

619. 

14

2) Jajcanin-Jozic, N., Deller, S., Pavkov, T., Macheroux, P., Abramic, M.: Identification of 

the reactive cysteine residues in yeast dipeptidyl peptidase III, Biochimie, 2010, 92:89- 

96. 

Award 

Dissertation Award of the Austrian Society for Molecular Biosciences and Biotechnology 

(ÖGMBT) to Andreas Winkler for his dissertation on “Structure-function studies on 

berberine bridge enzyme (BBE) from the California poppy, Eschscholzia californica”. Dr. 

Andreas Winkler was a PhD student in the Doktoratskolleg “Molecular Enzymology” and is 

now a postdoctoral fellow at the Max-Planck Institute for Molecular Medicine in Heidelberg, 

Germany. 

15

Cell Biology Group 

Group leader: Günther Daum 

Postdoctoral Fellow: Karlheinz Grillitsch (since May 2010) 

PhD students: Melanie Connerth, Sona Rajakumari, Karlheinz Grillitsch (till March 2010), 

Miroslava Spanova, Susanne Horvath, Martina Gsell, Vid V. Flis, Vasyl’ Ivashov, Lisa 

Klug 

Master students: Brigitte Wagner, Gerald Mascher 

Technicians: Claudia Hrastnik, Alma Ljubijankic (since November 2010) 


Functional organelles are 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 within the cell. To study biogenesis and 

maintenance of biological membranes and assembly of lipids into organelle membranes our 

laboratory makes use of the yeast as a well established experimental system. We combine 

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

lipid depot formation and membrane biogenesis. 

Specific aspects studied recently in our laboratory are (i) assembly and homeostasis of 

phosphatidylethanolamine in yeast organelle membranes with emphasis on the role of the 

major phosphatidylethanolamine synthesizing enzyme, the phosphatidylserine decarboxylase 

1, (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 

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

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

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

(ii) decarboxylation of PS 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 and Tgl3p. To obtain more insight into biosynthesis, assembly and homeostasis of 

PE, 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 PE homeostasis of the yeast Saccharomyces 

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

global view of the role of PE in the cell and to study the effects of an unbalanced PE level we 

subjected a psd1∆ deletion mutant and the corresponding wild type to DNA microarray 

analysis and examined genome-wide changes in gene expression. Comparison of the gene 

expression pattern of the psd1∆ mutant with the wild type led to the identification of ~50 

differentially expressed genes. Grouping of these genes into functional categories revealed 

that PE formation by Psd1p influenced the expression of genes involved in diverse cellular 

pathways including transport, carbohydrate metabolism and stress response. Currently, the 

16

most promising candidates of this screening are under investigation. This study will provide 

novel evidence for the complex network of phospholipid synthesis in the yeast. 

To understand cellular PE homeostasis in more detail, we also performed experiments 

defining traffic routes of PE within the yeast cell. In recent studies, we investigated the 

plasma membrane as destination for PE traffic. We employed yeast mutants bearing defects 

in the different pathways of PE synthesis and demonstrated that PE formed through all four 

pathways can be supplied to the plasma membrane. The fatty acid composition of 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 PE 

species. We assume that the phospholipid composition of 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. 

Psd1-precursor 

OM 

Tom 

40 

Tom 

IMS 

 

Psd1p-mature 

IM 

 

TIM23 

Oct 

1 

MP 

P 

Import of Psd1p into mitochondria 

A central aspect of this project is characterization of Psd1p regarding its molecular 

properties. Like most mitochondrial proteins, Psd1p is synthesized on free cytosolic 

ribosomes and imported into mitochondria where processing occurs. The Psd1-proenzyme 

contains a mitochondrial targeting sequence, an internal sorting sequence, and an alpha- and 

a beta-subunit which are linked through an LGST cleavage site. Cleavage at this site leads to 

the mature and active form of the enzyme generating a pyruvoyl group at the N-terminus of 

the alpha subunit. In recent studies performed in collaboration with the laboratory of Prof. N. 

Pfanner, Freiburg, Germany, we investigated i) the precise import route of Psd1p through the 

mitochondrial membranes, ii) the specific role of the LGST cleavage site on the import, 

assembly and maturation of the enzyme, (iii) the topology of Psd1p in the inner 

mitochondrial membrane; iv) the effect of mitochondrial processing peptidases on protein 

maturation, and v) possible complex formation of mature Psd1p. 

The link between PE metabolism and peroxisome proliferation is subject to another current 

investigation with emphasis on the role of enzymes and lipid transport routes involved. 

Previous studies suggested that PE formed through all four pathways (see above) and in 

different subcellular membranes can be supplied to peroxisomes with comparable efficiency. 

17

However, mechanisms involved in these translocation processes are still unclear. To address 

these questions, we established in vitro and in vivo assays for studying phospholipid supply 

to peroxisomal membranes. We constructed a strain which lacks the gene product of OPI3, 

the major PE methyltransferase localized to the ER, and bears an Opi3p-GFP hybrid with an 

SKL targeting sequence that directs the enzyme to peroxisomes. In this “reporter mutant”, 

the only site of phosphatidylcholine (PC) formation via methylation of PE are the 

peroxisomes, and the appearance of PC becomes an indicator and measure for PE 

translocation from the different sites of synthesis to peroxisomes. Currently permeabilized 

cells of the “reporter mutant” are used to characterize the PE transport to peroxisomes in 

some detail. This system in combination with mutations in the different PE biosynthetic 

pathways will allow us to investigate the different mechanisms of PE translocation between 

organelles involved in aminoglycerophospholipid biosynthesis. 

Most recently, we also studied the link between PE metabolism and neutral lipid storage. For 

this purpose we analyzed lipids from strains bearing defects in PE synthesis. These analyses 

showed that a mutant bearing a defect in the CDP-ethanolamine pathway had a decreased 

level of triacylglycerols (TAG). In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDPethanolamine 

pathway both the cellular and the microsomal levels of PE were markedly 

decreased, whereas in other mutants of PE biosynthetic routes depletion of this 

aminoglycerophospholipid in microsomes was less pronounced. This observation is 

important because the TAG synthesisizing enzyme Lro1p similar to the enzymes of the CDPethanolamine 

pathway is a component of the ER. We conclude from these results that in 

cki1∆dpl1∆eki1∆ insufficient local supply of PE to Lro1p was a major reason for the strongly 

reduced TAG level. 

Neutral lipid storage in lipid particles and mobilization 

Yeast cells have the capacity to store neutral lipids TAG and STE (steryl esters) in subcellular 

structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized 

and serve as building blocks for membrane biosynthesis. In a long-standing project of our 

laboratory, we investigate the characterization of enzymatic steps which lead to the 

mobilization of TAG and STE depots. 

A major focus of our neutral lipid project was the biochemical characterization of the three 

yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had 

demonstrated that deletion of TGL3 encoding the major yeast TAG lipase resulted in 

decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids, 

especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG 

fraction. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we 

carried out biochemical experiments with wild type and deletion strains bearing defects in 

Tgl3p, Tgl4p and Tgl5p. We demonstrated that tgl mutants had a lower level of sphingolipids 

and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG 

accumulation in these mutant cells resulted in reduced amounts of phospholipids and 

sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected 

the metabolic flux of long chain fatty acids and very long chain fatty acids required for 

sphingolipid and glycerophospholipid synthesis. The pattern of phosphatidylcholine (PC), PE 

and PS molecular species was altered in tgl deletion strains underlining the important role of 

TAG turnover in maintenance of the pool size and remodelling of complex membrane lipids. 

This study shed new light on the physiological role of TAG lipases in yeast and in general. 

18

Even more surprising evidence was obtained when the enzymology of the three TAG lipases 

Tgl3p, Tgl4p and Tgl5p was studied in some detail. Motif search analysis indicated that Tgl3p 

and Tgl5p did not only contain the TAG lipase but also an acyltransferase motif. Interestingly, 

lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of 

TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. 

Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both 

enzymes did not only exhibit lipase activity but also catalyzed acylation of 

lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using 

variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two 

enzymatic activities act independently of each other. These results demonstrated that yeast 

Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and 

catabolic processes. 

P-Lipase 

28 GGGTFG 29 

Acyltransferase 

48 HIIACQ 49 

1aa 

282 Patatin domain 48 

3 

910aa 

31 GSSAG 31 

Lipase 

Domains of Tgl3p (from Rajakumari and Daum, 2010, J. Biol. Chem. 285, 15769-15776) 

In another study, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of 

adipose TAG lipase (ATGL), catalyzes multiple functions in lipid metabolism. An extended 

domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus 

domain but also a conserved motif for calcium independent phospholipases A2 (PLA2). We 

showed that Tgl4p exhibits TAG lipase, STE hydrolase and PLA2 activities, but also 

catalyzes acyl-CoA dependent acylation of lysophosphatidic acid (LPA) to phosphatidic acid. 

Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and 

specifically phosphatidic acid synthesis. Moreover, deletion of TGL4 in Saccharomyces 

cerevisiae showed an altered pattern of PC and phosphatidic acid molecular species. 

Altogether, our data suggested that yeast Tgl4p functions as hydrolytic enzyme in lipid 

degradation, but also contributes to fatty acid channelling and phospholipid remodelling. 

Several years ago we identified through a mass spectrometric approach for the first time the 

major lipid particle proteins of Saccharomyces cerevisiae. This approach was a milestone in 

the field because it identified a number of new gene products and their function and provided 

valuable hints for processes associated with lipid particles. Recently, a more precise lipid 

particle proteome analysis was initiated in collaboration with M. Karas from the Institute of 

Pharmaceutical Chemistry, Johann Wolfgang Goethe University, Frankfurt, Germany. This 

proteome study was combined with a lipidomics investigation that was performed in 

collaboration with H. Köfeler from the Center for Medical Research, Medical University of 

Graz, Austria. In this study, we compared lipid particle components from cells which were 

19

grown on glucose or on oleic acid. This approach identified a number of lipid particle proteins 

that were already known but also some novel polypeptide candidates. We also realized 

through this approach that there were some differences in the lipid particle proteome from 

cells grown on glucose or oleic acid. Finally, mass spectrometric analyses revealed marked 

differences in the lipidome of lipid particles from cells grown on the two different carbon 

sources. This study sets the stage for further investigation of protein-lipid interaction on the 

surface of lipid particles and provides basic evidence for the coordinated biosynthesis of lipid 

and protein components from lipid particles. 

Another important aspect of this project was to understand cell biological consequences of 

dysfunctions in non-polar lipid storage. For this purpose, yeast cells were cultivated on oleic 

acid which was assumed to provoke lipotoxic stress. We found that under these cultivation 

conditions TAG synthesis was enhanced creating the major pool for the excess of fatty acids, 

whereas surprisingly STE synthesis was strongly inhibited. We showed that this effect was 

not due to decreased expression of ARE2 encoding the major yeast STE synthase at the 

transcriptional level, but to competitive enzymatic inhibition of Are2p by free oleic acid. As a 

result, a triple mutant dga1∆lro1∆are1∆ARE2 + grown on oleate did not form substantial 

amounts of STE and exhibited a growth phenotype similar to the dga1∆lro1∆are1∆are2∆ 

quadruple mutant which lacks all four acyltransferases involved in neutral lipid synthesis and 

consequently also lacks lipid particles. Growth of these mutants on oleate was strongly 

delayed and cell viability was decreased, but rescued by an adaptation process. In these 

strains, oleate stress caused morphological changes of intracellular membranes, altered 

phospholipid composition and increased formation of ethyl esters as a possible buffer for fatty 

acids. 

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. In a previous 

study we had demonstrated that squalene accumulates in yeast strains bearing a deletion of the 

HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets 

together with TAG and STE. In mutants lacking the ability to form lipid particles, however, 

substantial amounts of squalene accumulate in organelle membranes. In a recent study, we 

investigated the effect of squalene on biophysical properties of lipid particles and membranes 

and compared these results to artificial membranes. Our experiments showed that squalene 

lowered the order of STE shells in lipid particles. The majority of squalene, however, was 

localized to the center of lipid particles where it formed a soft core together with TAG. This 

view was confirmed with model lipid particles. In biological and artificial membranes 

fluorescence spectroscopy studies revealed that it is not the absolute squalene level per se, but 

the squalene to ergosterol ratio which mainly affects membrane fluidity/rigidity. In a fluid 

membrane environment squalene induces rigidity of the membrane, whereas in rigid 

membrane there is almost no additive effect of squalene. Our results demonstrated that 

squalene (i) can be well accommodated in yeast lipid particles and organelle membranes 

without causing deleterious effects; and (ii) although not being a typical membrane lipid may 

be regarded as a mild modulator of biophysical membrane properties. 

Pichia pastoris organelles and lipids 

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. Recent work 

20

focused on the biochemical characterization of the plasma membrane and secretory organelles 

from Pichia pastoris. One major aim of this project is the qualitative and quantitative analysis 

of phospholipids, fatty acids and sterols from organelle membranes when Pichia pastoris is 

grown on different carbon sources. For this purpose, methods for the isolation of Pichia 

pastoris organelle fractions were established. Standardized techniques of lipid analysis 

including lipidome analyses are currently employed to address these problems. 

Doctoral Theses completed 

Sona Rajakumari: Role of yeast triacylglycerol lipases in membrane lipid metabolism 

Previous work from our laboratory had demonstrated that gene products of TGL3, TGL4 and 

TGL5 encoding the major yeast triacylglycerol (TAG) lipases are located to lipid particles. 

Deletion of TGL3 and TGL4 resulted in a decreased mobilization of TAG from the lipid 

particles and a sporulation defect. TAG stored in tgl3∆ and tgl5∆ deletion strains contains 

slightly increased amounts of C22:0 and C26:0 very long chain fatty acids (VLCFAs) 

compared to wild type. These VLCFAs are indispensable for sphingolipid biosynthesis and 

crucial for raft association in yeast. Moreover, deletion of these TAG lipases results in a 

decreased level of membrane lipid biosynthesis. In this Thesis the role of TAG lipases in 

membrane lipid metabolism of the yeast Saccharomyces cerevisiae was studied in some 

detail. First, a metabolic link between TAG lipolysis by TAG lipases and sphingolipid and 

phospholipid synthesis was shown. Secondly, a dual function of Tgl3p and Tgl5p as lipases 

and as lysophosphatidylethanolamine or lysophosphatidic acid acyltransferase, respectively, 

was demonstrated. We also showed that the acyltransferase but not the lipase function of 

Tgl3p was essential for efficient sporulation. Finally, we also characterized Tgl4p as 

multifunctional protein exhibiting lipase, phospholipase A2, acyl-CoA dependent LPA 

acyltransferase and STE hydrolase activity. Altogether, this work shows that catabolic and 

anabolic activities of TAG lipases play a pivotal role in maintaining lipid homeostasis in the 

yeast. 

Karlheinz Grillitsch: Lipid storage and mobilization in the yeast Saccharomyces cerevisiae 

The yeast Saccharomyces cerevisiae like higher eukaryotic cells (mammals and plants) and 

Gram-positive bacteria contains a specified organelle for lipid storage, the lipid particle (LP). 

Unlike other organelles, LP are covered by a phospholipid monolayer that protects its 

hydrophobic interior formed from densely packed non-polar lipids steryl esters (STE) and 

triacylglycerols (TAG). Moreover, LP contain a small but specific set of proteins. In this 

Thesis, storage and mobilization of yeast neutral lipids were studied. First, biochemical 

properties of the three STE hydolases, Tgl1p, Yeh1p and Yeh2p were investigated. Analysis 

of enzymatic properties revealed distinct substrate specificities of the three proteins and 

involvement in sterol homeostasis. Sterol homeostasis is also linked to cell polarity. We 

showed that two effectors of cell polarity, Ste20p and Cla4p, function as negative modulators 

of sterol biosynthesis. A major part of this Thesis was devoted to description of the molecular 

composition of the yeast LP and its modulation upon changes in cultivation conditions. For 

this purpose, LP from cells grown on either glucose or oleate were analyzed. Strong 

incorporation of the mono-unsaturated oleic acid into TAG and most phospholipids was 

observed upon shifting cells to oleate medium. Most notably, the balanced 1:1 ratio of TAG to 

STE in cells grown on glucose was strongly increased on oleate. Change of the medium also 

led to changes in the LP protein pattern. This was demonstrated by a combined 

21

lipidomic/proteomic approach which also revealed several novel putative LP proteins. Finally, 

LP protein targeting and topology were studied using the squalene epoxidase Erg1p, a typical 

LP protein, as a model. These studies showed that the majority of the protein faced the 

cytosol, and only a small part was protected by the membrane. We assume that this may be a 

general feature of LP proteins. 

Melanie Connerth: Lipid traffic in the yeast 

The yeast Saccharomyces cerevisiae is a suitable organism for studies of lipid homeostasis in 

the cell. Processes involved in the biosynthesis of lipid compounds such as fatty acids, neutral 

lipids and phospholipids are well understood and located to different subcellular 

compartments. Two major yeast organelles involved in fatty acid turnover are peroxisomes 

harboring steps of beta-oxidation and lipid particles (LP) which are the storage organelle for 

excess fatty acids in the cell. Biogenesis of both compartments is not completely understood 

yet and little is known about mechanisms of lipid supply to either of these organelles. This 

Thesis was aimed at elucidation of lipid traffic routes towards and from peroxisomes and LP 

and the interplay of fatty acid utilization processes in both compartments. Initially, 

phospholipid supply to peroxisomal membranes was investigated in some detail with focus on 

phosphatidylethanolamine (PE) transport to peroxisomes. These studies revealed that the four 

different PE biosynthetic pathways contributed with different efficiency to this process. 

Moreover, a hybrid of the PE methyltransferase Opi3p was introduced into peroxisomes of an 

OPI3 deletion mutant which allowed us to measure transport of PE to peroxisomes by 

appearance of phosphatidylcholine (PC) in peroxisomal membranes. The major take-home 

message from these experiments was that direct membrane contact most likely accounts for 

delivery of lipids from their sites of synthesis to peroxisomes. During these studies, a link of 

peroxisomes with LP, the neutral lipid storage compartment, was also discovered. It was 

shown that exogenous oleic acid was primarily stored in LP in the form of triacylglycerols 

(TAG) and subsequently mobilized by a subset of already known as well as novel 

lipases/hydrolases for supply to peroxisomes. In the course of complete lipidome and 

proteome analyses of LP a novel and specific inhibitory effect of oleate on the activity of the 

steryl ester synthase Are2p was discovered thus revealing a new aspect of yeast lipotoxicity. 

In summary, data from this Thesis showed that distinct pools of fatty acids serving for 

different cellular processes exist in the cell, and LP play a major role in regulating lipid 

homeostasis. These data are a step forward in our understanding of cellular lipid traffic which 

may also be applicable to higher eukaryotic cells such as plants and mammals. 


N. Pfanner, Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg, 

Germany 

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 

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

Frankfurt, Germany 

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

22


FWF P21429: Phosphatidylserine decarboxylase 

FWF P18857: 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 

FWF P23029 Lipases of the yeast Saccharomyces cerevisiae 

Austrian Center of Industrial Biotechnology (ACIB): Pichia pastoris Cell factory and Protein 

Production 

Invited Lecture 

1. S. Rajakumari, M. Connerth, K. Grillitsch, S. Horvath, R. Rajasekharan and G. Daum 

Fatty acid channeling from depots to biomembranes in the yeast 

International Lipid Symposium: Cell Biology and Metabolism, Beijing, China, 1-3 

September 2010 

Publications 

1. Rajakumari, S. and Daum, G. 

Janus-faced enzymes yeast Tgl3p and Tgl5p catalyze lipase and acyltransferase 

reactions 

Mol. Biol. Cell 21 (2010) 501-510 

2. Schuiki, I., Schnabl, M., Czabany, T., Hrastnik, C. and Daum, G. 

Phosphatidylethanolamine synthesized by four different pathways is supplied to the 

plasma membrane of the yeast Saccharomyces cerevisiae. 

Biochim. Biophys. Acta 1801 (2010) 480-486 

3. Spanova, M., Czabany, T., Zellnig, G., Leitner, E., Hapala, I. and Daum, G. 

Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast 

Saccharomyces cerevisiae. 

J. Biol. Chem. 285 (2010) 6127-6133 

4. Rajakumari, S. and Daum, G. 

Multiple functions as lipase, steryl ester hydrolase, phospholipase and acyltransferase of 

Tgl4p from the yeast Saccharomyces cerevisiae. 

J. Biol. Chem. 285 (2010) 15769-15776 

5. Wagner, M., Hoppe, K., Czabany, T., Heilmann, M., Daum, G., Feussner, I. and Fulda, 

M. 

Identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 2 

(DGAT2) gene from the microalga O. tauri. 

Plant Physiol. Biochem. 48 (2010) 407-416 

6. Connerth, M., Czabany, T., Wagner, A., Zellnig, G., Leitner, E., Steyrer, E. and Daum, 

G. 

Oleate inhibits steryl ester synthesis and causes liposensitivity in the yeast. 

J. Biol. Chem. 285 (2010) 26832–26841 

7. Rajakumari, S, Rajasekharan, R. and Daum, G. 

Triacylglycerol lipolysis is linked to sphingolipid and phospholipid metabolism of the 

yeast Saccharomyces cerevisiae. 

Biochim. Biophys. Acta 1801 (2010) 1314-1322 

23

Molecular Biology Group 

Group leader: Karin Athenstaedt 

Postdoctoral Fellow: Andreas Beranek 

Acyltransferases catalyzing triacylglycerol synthesis in the oleaginous yeast Yarrowia 

lipolytica 

The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to accumulate huge 

amounts of triacylglycerols (TAG). Along with other neutral lipids, TAG are stored in socalled 

lipid particles, cell compartments with a rather simple structure. A neutral lipid core 

mainly formed of TAG and steryl esters is surrounded by a phospholipid monolayer with few 

proteins embedded. By growing Yarrowia lipolytica cells in media containing, e.g., industrial 

fats or glycerol as a carbon source, the amount of TAG can be increased up to 40% of cell dry 

weight. This ability 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, we started to investigate the proteome 

of Yarrowia lipolytica for proteins involved in TAG synthesis. Homology searches with TAG 

synthases of other eukaryotes as queries highlighted two candidate gene-products of the 

oleaginous yeast potentially catalyzing the formation of TAG. 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 (Fig. 1). 

A B 

C 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. 

In contrast to the negative control (B; mutant + empty plasmid), lipid particles are formed in 

mutant cells transformed with plasmids bearing either of the respective candidate genes of 

Yarrowia lipolytica (C, D). Panel A shows lipid particle formation in a wild-type cell of 

Saccharomyces cerevisiae. Lipid particles are indicated by arrows. Size bar: 10 µm. 

Fluorescent microscopic inspection and lipid analyses of the respective mutants clearly 

demonstrated that these Yarrowia lipolytica genes encode true TAG synthases. The 

24

characteristics of these two TAG synthases of the oleaginous yeast are currently under 

investigation. 

Biosynthesis of phosphatidic acid in yeast 

Phosphatidic acid is of utmost importance, since it is the key intermediate for the formation of 

all glycerolipids, namely glycerophospholipids (membrane lipids) and triacylglycerols 

(storage lipids). Furthermore, this lipid functions in cell signaling. In all eukaryotic organisms 

enzymes catalyzing phosphatidic acid biosynthesis occur in redundancy. One reason for the 

existence of isoenzymes may be the formation of different phosphatidic acid pools supplying 

different pathways. In this project this hypothesis is tested in the model organism yeast 

Saccharomyces cerevisiae, which contains two glycerol-3-phosphate acyltransferases, Gat1p 

and Gat2p, and two 1-acyl glycerol-3-phosphate acyltransferases, Slc1p and Lpt1p. Glycerol- 

3-phosphate acyltransferases catalyze the first and rate limiting step in de novo synthesis of 

phosphatidic acid via the glycerol-3-phosphate pathway, namely the acylation of glycerol-3- 

phosphate yielding 1-acyl glycerol-3-phosphate (lyso-phosphatidic acid). A subsequent 

acylation converts lyso-phosphatidic acid to phosphatidic acid and is catalyzed by a 1-acyl 

glycerol-3-phosphate acyltransferase. 

The precise localization of glycerol-3-phosphate acyltransferases tagged with green 

fluorescent protein (GFP) has been determined by fluorescence microscopy and Western blot 

analyses with highly purified cell fractions of the respective mutants. Whereas Gat1p is 

associated with lipid particles and the endoplasmic reticulum, Gat2p is exclusively localized 

to the latter compartment in wild-type background (Fig. 2). 

A 

B 

LP 

LP 

Wild-type + GFP-Gat1p 

Wild-type + GFP-Gat2p 

Figure 2: Fluorescence microscopy of Gat1p 

and Gat2p, respectively, tagged with green 

fluorescent protein (GFP) in wild-type 

background. 

A: GFP-Gat1p localizes to the endoplasmic 

reticulum and lipid particles (indicated by 

arrows). In contrast, GFP-Gat2p is only present 

in the endoplasmic reticulum (B). 

Most interestingly, the absence of either glycerol-3-phosphate acyltransferase results in a 

different distribution pattern of its counterpart, and in growth defects. Whether these 

phenotypes are caused by alterations in the lipid pattern of the respective mutants is currently 

under investigation. 


T. Chardot, Institut National de la Recherche Agronomique, Institut Jean-Pierre Bourgin, 

UMR1318, Versailles, France 

J.-M. Nicaud, Institut National de la Recherche Agronomique, Laboratoire de Microbiologie 

et Génétique Moléculaire, UMR1319, Jouy-en-Josas, France 

25


FWF P21251: Biosynthesis of phosphatidic acid in yeast 

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, 2010, 471-480. 


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. 



26

Biophysical chemistry group 

Group leader: Albin Hermetter 

Postdoctoral Fellow: Heidemarie Ehammer 

PhD students: Ute Stemmer, Daniel Koller, Claudia Ramprecht, Lingaraju Marlingapla 

Halasiddappa, Bojana Stojcic 

Master students: Andreas Gasser, Gabriel Pürstinger, Hannah Jaritz 

Technicians: Elfriede Zenzmaier 


Our research deals with the role of glycero(phospho)lipids and lipid modifying enzymes as 

components of membranes and lipoproteins, their function 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 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 

OXPHOS-EuroMEMBRANE 

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 consortia 

LIPOTOX (SFB) and OXPHOS (EuroMEMBRANE), we investigate the “Toxicity of 

oxidized phospholipids in macrophages” and “The protein targets and apoptotic signaling of 

oxidized phospholipids”. These studies aim at identifying the molecular and cellular effects of 

an important subfamily of the oxidized phospholipids containing long hydrocarbon chains in 

27

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 

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 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 phospholipases are the subject of our studies in the 

framework of the joint research program GOLD-Genomics of Lipid-associated Disorders at 

KFU 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. 

28

2.1. Fluorescent suicide inhibitors for in-gel analysis of lipases and phospholipases 

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 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). 

Green: wt Red: ko Red: wt Green: ko 

DABGE Analysis of 

lipases and esterases 

in mouse adipose tissue 

of wt and lipase ko 

mice 

Differential activity-based gel electrophoresis (DABGE) was developed for comparative 

analysis of two lipolytic proteomes in one polyacrylamide gel. For this purpose, the active 

lipases/esterases of two different samples are labelled with fluorescent inhibitors that possess 

identical substrate analogous structures but carry different cyanine dyes as reporter 

fluorophores. After sample mixing and protein separation by 1-D or 2-D PAGE, the enzymes 

carrying the sample-specific colors are detected and quantified. This technique can be used 

29

for the determination of differences in enzyme patterns, e.g. due to effects of genetic 

background, environment, metabolic state and disease. 

Master Theses completed: 

Andreas Gasser: Determination of substrate- and stereoselectivity of lipases 

The elucidation of the reaction mechanisms of lipolytic enzymes requires the determination of 

the substrate- and stereoselectivities of these proteins. In order to find a more straightforward 

alternative to the common assays using radioactive substrates, a method using chiral, pyrenelabelled 

substrates was developed in this study, which allows the determination of substrateand 

stereoselectivity of lipases. This technique avoids the application of radioactive isotopes 

and as a consequence time consuming procedures. It is the basis for a simple and fast 

determination of lipase activity in high throughput analysis. On the basis of the robust and 

well characterized fungal Rhizomucor miehei lipase, the fluorescent substrates were tested for 

their applicability to determine substrate- and stereoselectivity of lipolytic enzymes using 

simple thin-layer chromatography. In order to obtain quantitative results and higher 

throughput, the method was adapted to HPLC analysis. This version was used to analyze 

activities as well as e.e. values of Rhizomucor miehei lipase, Chromobacterium viscosum 

lipase and Candida cylindracea cholesterol esterase. After overexpression in COS-7 cells, the 

selectivity and activity of the animal lipases adipose triglyceride lipase (ATGL), hormone 

sensitive lipase (HSL) and monoglyceride lipase (MGL) were measured in total cell lysates. 

Lipolytic activities were also determined in adipose tissue homogenates of ATGL- and HSLdeficient 

mice and compared to the wild type animals. The deficiency in the respective lipases 

correlated with the degradation patterns of the fluorescent substrates as determined by the 

above described method. 

Franziska Vogl: Transfection of macrophages with siRNA and plasmids. Expression and 

silencing of proteins targeted by oxidized phospholipids 

Oxidized phospholipids (oxPL) are generated from (poly)unsaturated glycerophospholipids in 

membranes and lipoproteins under conditions of oxidative stress. These compounds induce 

apoptosis in the cells of the vascular wall. In a previous functional proteomics study, the 

primary protein targets of a toxic aldehydophospholipid were identified in macrophages. To 

find out which of these protein candidates are involved in lipid-mediated cell death, we are 

following two different strategies. The first one uses siRNA technology to knock down the 

target proteins and measure the effect on cell death and signalling. The second approach is 

based on fusion proteins containing the protein candidates and RFP to measure Försterresonance-energy-transfer 

(FRET) and determine the spatial proximity to fluorescently 

labelled oxPL. In this diploma thesis, protocols for chemical transfection of RAW 264.7 cells 

with expression plasmids were optimized. Five fluorescent fusion proteins were cloned and 

expressed in Cos-7 cells. Chemical transfection and electroporation were used to transfect 

cells with siRNA against GAPDH and Caspase-3. Transfections and silencing were monitored 

using FACS-measurements, Western blotting and qPCR and proved to be efficient in both 

cases. Silencing of caspase-3 was associated with a decrease in protein expression and loss of 

function. As a consequence, enzyme knock-down rendered the cells more resistant towards 

oxPL-induced apoptosis, supporting the assumption that the protease is causally involved in 

programmed cell death. 

30

International cooperations: 

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

Slovakia 

T. Hornemann, Institute for Clinical Chemistry, University Hospital Zürich; Zürich, 

Switzerland 

M. Hof, Department of Biophysical Chemistry, Academy of Sciences of the Czech Republic, 

Prague 

D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine; 

Cornell University, Ithaca, USA 

I. Parmryd, Cell Biology, The Wenner-Gren Institute, Stockholm University, Stockholm, 

Sweden 

T. Hugel, IMETUM, TU München, Garching, Germany 

P. Kinnunen, Biophysics, Aalto University, Helsinki, Finland 

Research projects: 

BM.W_F Genomforschung in Österreich (GEN-AU): Exploring the lipolytic proteome. 

Phospholipases (GOLD 3 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. 

FWF EUROCORES-EuroMEMBRANE-OXPHOS-Protein targets and apoptotic signaling of 

oxidized phospholipids 

Invited lectures: 

1. Functional proteomic analysis of hydrolytic enzymes relevant to the stability and 

activity of oxidized phospholipids in macrophages. Cellular radical stress and related 

biomarkers, COST Chemistry CM0603, Athens, Greece, February 22, 2010 

2. Molecular and supramolecular targets of oxidized phospholipids. ESF Euromembrane 

Meeting, Heidelberg, Germany, April 7, 2010 

3. Primary targets of oxidized phospholipids in macrophages. 5 th Prague- Wroclaw 

Seminar on Biophysics, Prague, Czech Republic, November 18, 2010 

Publications: 

1. Landre JB, Blaess MF, Kohl M, Schlicksbier T, Ruryk A, Kinscherf R, Claus RA, 

Hermetter A, Keller M, Bauer M, Deigner HP 

Addressable Bipartite Molecular Hook (ABMH): Immobilized Hairpin Probes with 

Sensitivity below 50 Femtomolar 

Anal.Biochem. 397, 60-66, 2010. 

2. Sattler W, Nusshold C, Kollroser M, Köfeler H, rechberger G, reicher H, Üllen A, 

Bernhart E,Waltl S,Kratzer I,Hermetter A,Hackl H, Trajanoski Z,Hrzenjak A, Malle E 

Hypochlorite modification of sphingomyelin generates chlorinated lipid species that 

induce apoptosis and proteome alterations in dopaminergic PC12neurons in vitro 

Free Radical Biol Med 48, 1588-1600, 2010. 

31

3. Vanderven BC, Hermetter A, Huang A, Maxfield FR, Russell DG, Yates RM 

Development of a novel, cell-based chemical screen to identify inhibitors of 

intraphagosomal lipolysis in macrophages 

Cytometry A 77, 751-760, 2010. 

4. Watschinger K, Keller MA, Golderer G, Hermann M, Maglione M, Sarg B, Lindner 

HH, Hermetter A, Werner-Felmayer G, konrat R, Hulo, N, Werner ER 

Identification of the gene encoding alkylglycerol monooxygenase defines a third class of 

tetrahydrobiopterin dependent enzymes 

PNAS, 107, 13672-13677, 2010. 

5. Plochberger B, Stockner T, Chiantia S, Brameshuber M, Weghuber J, Hermetter A, 

Schwille P, Schuetz GJ 

Cholesterol slows down the lateral mobility of an oxidized phospholipid in a supported 

lipid bilayer 

Langmuir, 26, 17322-17329, 2010. 

6. Schicher M, Morak M, Birner-Gruenberger R, Kayer H, Stojcic B, Rechberger G, 

Kollroser M, Hermetter A 

Functional proteomic analysis of lipases and esterases in cultured human adipocytes 

J. Proteome Res., 9, 6334-6344, 2010. 

32

Chemistry of Functional Foods 

Group leader: Michael Murkovic 

PhD students: Alam Zeb, Yuliana Reni Swasti 

Diploma students: Elham Fanaee Danesch, Tatjana Golubkova, Zivile Rocyte 

Technician: Alma Ljubijankic 

Honorary Professor: Klaus Günther, Forschungszentrum Jülich, Germany 

Visiting Professors: Evangelos Katzojannos, TEJ of Athens, Greece; Zhong Hayan, Central 

South University of Forestry and Technology, China 


Antioxidants have different functions depending on the location of action. Is it the protection 

of biological systems maintaining the integrity of the system or the protection of foods against 

oxidation leading to health threatening substances The exposure to oxidation products is 

either described as oxidative stress or the oxidized substances have an acute or chronic 

toxicity or are carcinogenic. The production of healthier and safer foods is of primary interest 

of this research group. 

The antioxidants of interest are polyphenols including anthocyanins and carotenoids. The 

evaluation of their occurrence in food and their behavior during processing and cooking is 

important especially when these substances are used as food additives. The safety evaluation 

of these compounds includes the evaluation of possible degradation products. 

Heating of food is a process that is normally done to improve the safety and digestibility and 

improve the sensory attributes like texture, color, and aroma. During the heating reactions 

occur that lead to the degradation of nutritive constituents like carbohydrates, proteins, amino 

acids and lipids. Some of the reaction products are contributing to the nice aroma, color, and 

texture of the prepared food and many of them are highly toxic and/or carcinogenic. However, 

these hazardous compounds occur in rather low concentrations being normally not acute 

toxic. The substances have a very diverse chemical background like heterocyclic amines, 

polycondensated aromatic compounds, acrylamide or furan derivatives. The aim of the 

research is to investigate the reaction mechanisms that lead to the formation of these 

hazardous compounds and establish strategies to mitigate the formation and thereby reducing 

the alimentary exposure. 

Carotenoids thermal oxidation in triacylglycerols 

β-Carotene is one of most important and widespread carotenoids present in food. It is an 

important source of provitamin A. It acts as antioxidant in biological systems including the 

complex system of lipids. We studied the oxidation of β-carotene in model triacylglycerol 

systems, comparable to most of the high oleic edible oil. An HPLC electrospray ionization 

mass spectrometric method was developed for the separation and identification of 

triacylglycerols and its oxidation products (Figure 1). β-Carotene was found to act as 

antioxidant at lower temperature like 90°C and 100°C, while oxidation of triacylglycerols was 

closely correlated with the oxidation of β-carotene after 8 hours of thermal oxidation at 110 

°C. The oxidized products of β-carotene were epoxides, peroxides and apocarotenals, 

separated and analyzed using C30 reversed phase HPLC-DAD and APCI-MS. We found that 

110 °C and 120 °C were the favorable temperatures for studying the oxidation reaction of 

carotenoids, while 110 °C, 120 °C and 130 °C were favorable for triacylglycerol oxidation. 

33

Trilinolein 

263 

288 

O 

O 

O 

O 

O 

O 

599 

896 

901 

1-Oleoyl-2-linoleoyl-3-linoleoyl glycerol 

288 

O 

O 

O 

O 

O 

O 

599 

601 

O 

O 

O 

O 

O 

898 

+ 

881 

O 

872 

+ 

903 

Triolein 

263 

O 

O 

O 

O 

O 

O 

1-Oleoyl-2-oleoyl-3-stearoyl glycerol 

+ 

883 

+ 

O 

O 

O 

O 

O 

O 

Stability of triacylglycerols decreases with the increased formation of the oxidation products 

of β-carotene. 

100 

1-Oleoyl-2-linoleoyl-3-oleoyl glycerol 

[ M + Na] 

+ 

905 

603 

[ M + ] + NH4 

100 OO + 

902 

80 

80 

Intensity 

60 

OL + 

601 

[ M + NH4] 

+ 

900 

Intensity 

60 

[ M + Na] 

+ 

907 

40 

20 

288 

263 

0 

200 300 400 500 600 700 800 900 

m/z 

OO + 

603 

[ M-O +H+ Li] 

+ 

874 

+ [ M + H] 

883 

40 

20 

[ M + H] 

0 

200 300 400 500 600 700 800 900 

m/z 

[ M- O+ H + Li] 

876 

100 

+ [ M + Na] 

100 

+ [ M + Na] 

100 

[ M + NH4 ] + 

909 

[ M + Na] 

+ 

904 

80 

Intensity 

80 

60 

40 

20 

[ M + NH 4] 

+ 

+ [ M + H] 

879 

0 

200 300 400 500 600 700 800 900 

m/z 

LL + 

Intensity 

80 

60 

40 

20 

LL + 

[ M + NH4] 

+ 

[ M-O+ Li] 

H+ 

[ M + H] 

0 

200 300 400 500 600 700 800 900 

m/z 

OL + 

Intensity 

60 

OO + 

603 

887 

40 

20 

288 [ M + H 

+ 

] 

0 

200 300 400 500 600 700 800 900 

OS + 

605 

DAGs 

HPLC-MS 

0 5 10 15 20 25 30 

Time (min) 

Figure 1. Separation and Identification of Triacylglycerols using HPLC-ESI-MS. 

Doctoral Thesis completed 

Alam Zeb: Carotenoids and Triacylglycerols Interactions during Oxidation 

Carotenoids (β-carotene and astaxanthin) were oxidized in high oleic model triacylglycerols 

(TAGs) and edible oils such as corn and olive oils. The main techniques used in this 

dissertation were HPTLC and HPLC coupled to DAD and mass spectrometry. The previous 

literature on the uses of TLC suggests that HPTLC have the potential to be the first choice in 

the analysis of carotenoids in foods. We also found that HPTLC is a useful tool in the study of 

degradation of β-carotene in model TAGs and edible oils. The isocratic HPLC-ESI-MS 

method was very useful for the fast screening and identification of TAGs in edible oils. We 

correctly identify and separated thirteen, fourteen, fifteen and sixteen TAGs in refined olive 

oil, rapeseed oil, corn oil and sunflower oil, respectively. The oxidation products of TAGs 

were also studied using this method. Epoxy epidioxides, hydroxy bis-hydroperoxides and 

epidioxy bishydroperoxides were identified as major oxidized compounds that have been 

identified for the first time in model TAGs and edible oils under similar conditions. Other 

triacylglycerols oxidized species were hydroxy hydroperoxides, mono-hydroperoxides, bishydroperoxides, 

epoxy-epidioxides, and epoxides. Significant degradation of β-carotene was 

observed in sunflower oil. In high oleic model TAGs, β-carotene degraded significantly in the 

first three hours, however, in olive oil of relatively similar TAG composition, β-carotene 

degraded slowly. Astaxanthin degradation was much slower than β-carotene in olive oil. The 

HPLC method for the degradation and oxidation of carotenoids reveal a total of eight oxidized 

compounds of β-carotene in corn oil. The degradation of all-E-β-carotene in corn oil was 

relatively similar to model TAGs and olive oil. The interactions of carotenoids and TAGs 

reveal the pro-oxidant action of both β-carotene and astaxanthin. The pro-oxidant action of β- 

carotene was much stronger than astaxanthin. These findings help us to understand the 

34

structural characterization of TAGs using mass spectrometry and the possible role and 

interactions of carotenoids or its oxidation products with the normal and oxidized 

triacylglycerols during thermal oxidation. 

Master Thesis completed 

Elham Fanaee Danesch: Ethanol production from fruit waste with solid state fermentation 

using Saccharomyces cerevisiae 

Disposal of agricultural wastes including rotten fruits and vegetables particularly in large 

cities and the lack of an appropriate method of recycling not only causes environmental 

pollution but also needs a loss of resources. These wastes are a good source of carbohydrates, 

acids, fibers, inorganic compounds and vitamins. Therefore they have a good potential for 

production of ethanol, animal feed, enzymes, pectin, etc. In this research, production of 

ethanol from fruit wastes by Saccharomyces cervisiae was investigated at 28 °C, pH 4.5 and 

77 % moisture in solid state fermentation. S. cervisiae was obtained from Biochemical and 

Bioenvironmental Research Center (B.B.R.C.). A maximum ethanol production yield of 13.75 

% based on the initial concentration was obtained from sugar after 22 h of fermentation which 

is equivalent to 1 g of ethanol per 42.35 g of fruit waste. The optimum value of effective 

parameters in production of ethanol were found to be 1 % ammonium sulfate, 1.5 % 

potassium dihydrogen phosphate, 2 % glucose and 41.06 g fruit waste. At the end of the 

fermentation process, 71 % of the substrate sugar was consumed. 


R. Venskutonis, Institute of Food Technology, Kaunas University of Technology, Lithuania 

T. Husoy, National Institute of Public Health, Olso, Norway 

H.R. Glatt, Deutsches Institute für Ernährungsforschung, Potsdam Rehbrücke, Germany 

E. Lazos, TEJ of Athens, Greece 

R. Grujic, University of East Sarajevo, Bosnia and Herzegovina 

E. Habul, University of Sarajevo, Bosnia and Herzegovina 

E. Winkelhausen, S. Kuzmanova, University Ss Cyril and Methodius, Skopie, FRYM 

H. Pinheiro, Instituto Superior Tecnico, Lisboa, Portugal 

V. Piironen, Department of Applied Chemistry and Microbiology, Helsinki, Finland 

M.J. Cantalejo, Department of Food Technol., Public University of Navarre, Pamplona, Spain 

Z. Cieserova, Food Research Institute, Bratislava, Slovakia 

C. Thongkraung, Prince of Songkla University, Hatyai, Thailand 

Research project 

European Network of Excellence: EuroFIR European Food Information Resource 

Lectures 

1) A. Zeb 

ß-Carotene induced oxidation of high oleic triacylglycerols model system. 

Österreichische Lebensmittelchemikertage. Schloss Seggau-Leibnitz, 19 May 2010 

35

2) M. Murkovic 

Zusatzstoffe - unverzichtbare Begleiter in der Küche 

Wissenschaft mit Geschmack. Graz, 16 June 2010 

3) A. Zeb 

Role of Astaxanthin in Thermal Oxidation of High Oleic Triacylglycerols. 

8 th Euro Fed Lipid Congress. Munich, 21 Nov. 2010 

Publications 

1) Zeb, A.; Murkovic, M.: 

Analysis of triacylglycerols in refined edible oils by isocratic HPLC-ESI-MS. 

European journal of lipid science and technology 112 (2010) 8, S. 844 – 851 

2) Jöbstl, D.; Husoy, T.; Alexander, J.; Bjellaas, T.; Leitner, E.; Murkovic, M.: 

Analysis of 5-hydroxymethyl-2-furoic acid (HMFA) the main metabolite of alimentary 

5-hydroxymethyl-2-furfural (HMF) with HPLC and GC in urine. 

Food Chemistry 123 (2010), S. 814 - 818 

3) Prasetyo, E. N.; Kudanga, T.; Steiner, W.; Murkovic, M.; Wonisch, W.; Nyanhongo, G. 

S.; Gübitz, G.: 

Cellular and plasma antioxidant activity assay using tetramethoxy. 

Free radical biology & medicine 49 (2010), S. 1205 - 1211 


Characterization of the effects of β-Carotene on the thermal oxidation of 

Triacylglycerols using HPLC-ESI-MS. 

European journal of lipid science and technology 112 (2010) 11, S. 1218 - 1228 


High-Performance Thin-Layer Chromatographic Method for Monitoring the Thermal 

Degradation of β-Carotene in Sunflower Oil. 

Journal of Planar Chromatography - modern TLC 23 (2010) 1, S. 35 - 39 

6) Nugroho Prasetyo, E.; Kudanga, T.; Steiner, W.; Murkovic, M.; Nyanhongo, G. S.; 

Gübitz, G.: 

Laccase-generated tetramethoxy azobismethylene quinone (TMAMQ) as a tool for 

antioxidant activity measurement. 

Food Chemistry 118 (2010) 2, S. 437 - 444 


Thin-Layer Chromatographic Analysis of Carotenoids in Plant and Animal Samples. 

Journal of Planar Chromatography - modern TLC 23 (2010) 2, S. 94 - 103 

Scientific Conference 

Biannual Conference of the Austrian Food Chemists 

Austrian Chemical Society – WG Food Chemistry, Cosmetics, and Tensides 

19.5. – 21.5.2010, Schloss Seggau, Leibnitz, Austria 

36

Lectures and Laboratory Courses 

Aktuelle Trends und Ergebnisse in der 

2 VO Günther K 

Analytischen Chemie und Lebensmittelchemie 

Anleitung zu wissenschaftlichen Arbeiten aus 

Biochemie und Molekularer Biomedizin 

2 SE Daum G, Hermetter A, 

Macheroux P 

Anleitung zu wissenschaftlichen Arbeiten aus 

Biochemie und Molekularer Biomedizin 

2 SE Daum G, Hermetter A, 

Macheroux P 

Bioanalytik 2.25 VO Hermetter A, Klimant I 

Biochemie I 3.75 VO Macheroux P 

Biochemie II 1.5 VO Macheroux P 

Biophysikalische Chemie 1 2 PV Laggner P 

Biophysikalische Chemie 2 2 PV Laggner P 

Biophysikalische Chemie der Lipide 1 2 PV Hermetter A 

Biophysikalische Chemie der Lipide 2 2 PV Hermetter A 

Chemie und Technologie der Lebensmittel II 2 VO Murkovic M 

Chemische Veränderungen in Lebensmitteln I 2 PV Murkovic M 

Chemische Veränderungen in Lebensmittel II 2 PV Murkovic M 

Chemische Veränderungen in Lebensmitteln 2 SE Murkovic M 

bei der Verarbeitung 

DissertantInnenseminar 1 1 SE Faculty 

DissertantInnenseminar 2 1 SE Faculty 

Enzymatische und mikrobielle Verfahren in der 2 VO Murkovic M 

Lebensmittelherstellung 

Fisch- und Fischprodukte 1 VO Murkovic M 

Fluoreszenztechnologie 2 VO Hermetter A 

Fluoreszenztechnologie 1.5 LU Hermetter A 

GL Biochemie (BMT) 2 VO Macheroux P, Waldner-Scott I 

GL Chemie (BMT) 2 VO Hermetter A 

GL der Pharmakologie 2 VO Dittrich P 

Immunologische Methoden 2 VO Daum G 

Immunologische Methoden 2 LU Binter A, Daum G, Knaus T, 

Wallner S 

Immunologische Methoden 1 VO Daum G 

Lebensmittelbiotechnologie 1.3 VO Murkovic M 

Lebensmittelchemie/Technologie 4 VU Leitner E, Murkovic M 

LU aus Biochemie I 5.33 LU Binter A, Daum G, Hermetter 

A, Knaus T, Macheroux P, 

Waldner-Scott I, Wallner S 

LU aus Biochemie II 4 LU Binter A, Daum G, Macheroux 

P, Waldner-Scott I, Wallner S 

LU aus Molekularbiologie 3 LU Knaus T, Waldner-Scott I 

Membran Biophysik 1 2 PV Lohner K 

Membran Biophysik 2 2 PV Lohner K 

Membran-Mimetik 1 VO Lohner K 

Molekulare Enzymologie 2 VO Gruber K, Macheroux P, 

Nidetzky B 

Molekulare Enzymologie I 2 PV Macheroux P 

Molekulare Enzymologie II 2 PV Macheroux P 

Molekulare Gastronomie und 1 EV Murkovic M 

37

Lebensmittelverarbeitung II 

Molekulare Physiologie 2 VO Macheroux P 

Physikalische Methoden in der Biochemie 2 LU Laggner P 

Physikalische Methoden in der Biochemie 1 VO Laggner P 

Projektlabor Bioanalytik 1 6 PR Daum G, Hermetter A, 

Macheroux P 

Projektlabor Bioanalytik 2 6 PR Daum G, Hermetter A, 

Macheroux P 

Projektlabor Biochemie und Molekulare 

Biomedizin 1 

Projektlabor Biochemie und Molekulare 

Biomedizin 2 

9 PR Trajanoski Z, Macheroux P, 

Daum G, Hermetter A, 

Waldner-Scott I 

9 PR Daum G, Hermetter A, 

Macheroux P, Trajanoski Z, 

Waldner-Scott I 

Projektlabor Lebensmittelbiotechnologie 1 6 PR Leitner E, Murkovic M 

Projektlabor Lebensmittelbiotechnologie 2 6 PR Leitner E, Murkovic M 

Seminar zu den LU aus Molekularbiologie 1 SE Athenstaedt K, Waldner-Scott I 

Spezielle Kapitel der Biochemie 1 VO Daum G, Hermetter A, 

Macheroux P 

Spezielle Kapitel der Lebensmittelchem.- u. 2 SE Murkovic M 

technologie I 

Spezielle Kapitel der Lebensmittelchemie- und 2 SE Murkovic M 

techn.II 

Strukturanalyse in Biophysik u. 

2 VO Laggner P 

Materialforschung 

Unterrichtspraxis 2 SE Faculty 

Unterrichtspraxis 2 SE Faculty 

Wissenschaftliches Kolloquium für 

1 SE Faculty 

DissertantInnen 1 

Wissenschaftliches Kolloquium für 

1 SE Faculty 

DissertantInnen 2 

Zellbiologie 1.5 VO Daum G 

Zellbiologie 1 1 SE Daum G 

Zellbiologie 2 1 SE Daum G 

Zellbiologie der Lipide 1 2 PV Daum G 

Zellbiologie der Lipide 2 2 PV Daum G 

38

Graz University of Technology Austria Institute of Biochemistry ...

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