Staff Members of the Institute of Biochemistry, TU - Institut für ...

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 

Dr. Alexandra Binter 

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

Dr. Ines Waldner-Scott 

(ines.waldner-scott@tugraz.at; Tel.:+43-(0)316-873-4522) 

DI Tanja Knaus 

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

DI 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 

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

1

Brief History of the Institute of Biochemistry 

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

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

Technology Graz. Together with all the other chemistry institutes, it was located in the old 

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

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

the Fürstlich-Dietrichstein-Stiftung, Schlögelgasse 9, in which all the biosciences were 

then concentrated. 

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

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

Biochemical Technology and Food Chemistry. 

1948 G. GORBACH was nominated full professor and head of the institute. In succession of 

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

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

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

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

2

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

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

docs in 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. Karin is 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. 

3

Highlights of 2011 

This was clearly the year for the flavin enthusiasts in the Macheroux group: the 

17 th International Symposium on Flavins and Flavoproteins was hosted by the University of 

California at Berkeley, USA, in July. Silvia Wallner, Thomas Bergner and Peter Macheroux 

presented posters revolving around “yellow enzymes”. 

4 

From left to right: 

Katharina Durchschein, Silvia 

Wallner, Thomas Bergner, 

Thomas Stoisser, Barbara Holl- 

Macheroux, Karl Gruber & 

Pauline Macheroux 

In September, Peter Macheroux was nominated as one of the top ten lecturers of Graz 

University of Technology. The official ceremony took place on November 8 th in the aula of 

the university. At the board meeting in October, the Austrian Science Fund (FWF) approved a 

new research project to investigate the mechanism of bacterial bioluminescence (Project 

P24189: “Bacterial bioluminescence”). Two former members of the group received 

fellowships to continue their scientific career: Dr. Andreas Winkler, currently a postdoctoral 

fellow at the Max-Planck Institute of Molecular Medicine in Heidelberg received an “Erwin- 

Schrödinger” stipend for 2012, and Dr. Sonja Sollner, currently at the Institute of Molecular 

Pathology in Vienna, was awarded a “Hertha-Firnberg” stipend. Both alumni were members 

of the PhD program “Molecular Enzymology”. Despite contracting budgets and stiff 

competition, this PhD program received excellent reviews and was extended for a second time 

until 2014. Three groups of the institute (Günther Daum, Albin Hermetter and Peter 

Macheroux) participate in this NAWI Graz interuniversity program. 

Norman Medal Award 

Ceremony at the 9 th Euro Fed 

Lipid Congress in Rotterdam, 

The Netherlands. Left K. Schurz, 

President of the DGF, right G. 

Daum. 

In 2011, three new PhD students 

joined the group of Günther 

Daum: Claudia Schmidt, 

Barbara Koch and Birgit Ploier 

started their studies on topics 

related to neutral lipid 

metabolism in the yeast. 

In 2011, Günther Daum started his third three year term as a Board Member of the Austrian 

Science Fund FWF. He also continued his work as President of the International Conference

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

Among the various invitations to research institutions and conferences a visit at the Central 

Institute of Medicinal and Aromatic Plants in Lucknow and to the Jawaharla Nehru 

University, Delhi, India, was definitely one highlight. In September 2011, Günther Daum 

received the Normann Medal, the highest Award of the Deutsche Gesellschaft für 

Fettwissenschaft (DGF), for his long term studies on biochemistry and cell biology of yeast 

lipids and biomembranes. 

Basic research in Albin Hermetter’s group focused on lipid 

(patho)physiology in human and animal cells. The respective 

projects were components of the research consortia GOLD/ GEN- 

AU (FFG), OXPHOS/ EuroMEMBRANE (ESF, FWF) and the PhD 

program “Molecular Enzymology” (FWF). A. Hermetter was 

invited to co-edit a special issue entitled “Oxidized phospholipidstheir 

properties and interactions with proteins” for Biochim. 

Biophys. Acta. Lingaraju Marlingapla Halasiddappa who is a PhD 

student in the PhD program “Molecular Enzymology” spent eight 

months in the Department of Biological Chemistry at Weizman 

Institute in Rehovot, Israel, to do research in the field of sphingolipid 

enzymology. He received an award for his oral presentation 

“Ceramide Synthases in Oxidized Phospholipid-induced RAW 

264.7 Macrophage Cell Death” at the International Charleston 

Ceramide Conference in Villars, Switzerland, March 2011. 

5 

Lingaraju Marlingapla 

Halasiddappa, 

PhD student 

In 2011 Karin Athenstaedt continued her work at our 

institute as an independent researcher. Her work is devoted to 

lipid metabolism in yeast. One of the highlights in Karin 

Athenstaedt’s scientific work in 2011 was attendance of the 

Gordon Conference (Molecular & Cellular Biology of Lipids) 

in Waterville Valley, NH, USA, where she had the 

opportunity to meet many experts in the field and to set the 

stage for collaborative activities. 

In the group Chemistry of Functional Foods directed by 

Michael Murkovic in 2011 a PhD thesis was ongoing in which 

the polymerization of furfuryl alcohol is investigated. Furfuryl 

alcohol is formed during heating of foods, e.g. during roasting 

of coffee. It can be activated metabolically which might result 

in the induction of tumors. The polymerization removes the 

furfuryl alcohol from the foods and reduces the bioavailability. 

In 2011 several international students (Lithuania, Egypt, 

Hungary, Slovak Republic) were coming to the lab for carrying 

out specific experiments related to, e.g. new technologies of 

heating, oxidation of oils in the bulk phase and in emulsions, 

and the application of asparaginase for reduction of the 

acrylamide formation.

Biochemistry Group 

Group leader: Peter Macheroux 

Secretary: Annemarie Portschy 

Senior scientists: Alexandra Binter, Ines Waldner-Scott 

PhD students: Thomas Bergner, Bastian Daniel, Venugopal Gudipati, Tanja Knaus, Wolf- 

Dieter Lienhart, Silvia Wallner 

Master students: Corinna Dully, Karin Koch, Julia Koop, Katharina Lukas, Nicole Sudi, 

Marlene Tösch 

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

Toukhy 

General description 

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

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

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

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

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

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

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

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

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

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

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

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

functional role in the 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 “berberine bridge”). The ultimate acceptor of the substratederived 

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

6

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 (PhD student) studies the functional role of several amino acid residues interacting 

with the isoalloxazine ring of the flavin cofactor. In addition, Silvia and Corinna Dully 

7

(master student) investigate whether alternative covalent modifications in the 8�-position are 

feasible, i.e. whether aspartate or tyrosine can form a covalent bond to the 8�-methyl group. 

In collaboration with Prof. Toni Kutchan at the Donald Danforth Plant Science Center in St. 

Louis, we have identified other plant genes that apparently encode flavin-dependent oxidases. 

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 Nterminus 

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 (PhD student in Karl Gruber’s group) 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 an inactive variant of human DPPIII with a 

tightly bound peptide. 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 

8

potentially useful inhibitors of the enzyme (this project is supported by Alexandra Binter in 

our group). 

Luciferase and LuxF 

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

in diverse organisms such as bacteria, fungi, 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) 

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 (R1= uracil & R2= OH, see below) is currently in clinical trial for its 

antifungal activity. Structurally, nikkomycins can be classified as peptidyl nucleosides 

containing two unusual amino acids, i.e. hydroxypyridylhomothreonine (HPHT) and 

aminohexuronic acid with an N-glycosidically linked base: 

9 

Structure of a LuxF dimer in the 

absence (red) and presence (blue) 

of the myristylated flavin 

derivative (pdb code 1NFP)

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. The biosynthesis commences with the attachment of an 

enolpyruvyl moiety to the 3´-position of UMP. Since none of the nikkomycins synthesized 

possess an enolpyruvyl group in this position of the sugar moiety, 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-Nacetylglucosamine 

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: 

Each 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/Rfree values of 17% and 19%, respectively. 

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 were cloned, expressed 

and the proteins purified for biochemical characterization and crystallization. Furthermore, 

nikkomycin intermediates produced by Streptomyces mutants featuring disruptions of the 

respective genes were analyzed, but no nikkomycins could be detected in their fermentation 

media. Comparisons of Nik enzyme sequences to those of known enzymes in the databases 

allow some predictions of the reactions each nik enzyme might catalyze. None of the enzymes 

was observed to convert 3´-EPUMP, the product of the NikO catalyzed reaction, so far. NikK 

10

is a pyridoxal-5-phosphate dependent aminotransferase, which is expected to introduce the 

amino group into the aminohexuronic acid moiety. Several amino acids were shown to serve 

as amino group donors, and L-glutamate was the most efficient one. NikJ possesses an ironsulfur 

cluster and probably belongs to the family of radical SAM enzymes. Its role in 

nikkomycin biosynthesis is unclear, but it is expected to function as an oxidoreductase. 

Cleavage of S-adenosylmethionine could not be observed. Furthermore, NikS was expressed, 

an enzyme that is expected to play a role in the assembly of nikkomycins (thesis project of 

Alexandra Binter and Gustav Oberdorfer in Karl Gruber’s laboratory, master thesis of Nicole 

Sudi). 

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 

Lot6p 

� 

� 

�� 

�� 

Yap4p 

Oxidation by 

e.g. 

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 p73�. Hence, the discovery of a homologous protein interaction in yeast provides an 

interesting model system to investigate the molecular basis for protein complex formation and 

regulation of proteasomal degradation of transcription factors (thesis project of Wolf-Dieter 

Lienhart and Venugopal Gudipati, master thesis of Karin Koch). 

11 

oxidized 

Lot6p 

Very slow with oxygen O2 nucleus 

oxygen 

�� 

� 

� 

�� 

Yap4p 

Yap4p

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 

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 2octylsulfate 

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 

Ala/Ile 

Tyr/Ser 

Met/Ser 

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). 

12 

Met/Ser 

Tyr/Ser 

Leu/Pro 

Ala/Ile

Doctoral thesis completed 

Alexandra Binter: Enzymes of nikkomycin biosynthesis 

Nikkomycins are peptide nucleosides which can be isolated from the fermentation broth of 

Streptomyces tendae and S. ansochromogenes. Due to their inhibition of chitin synthase they 

have fungicidal properties and a great potential as antibiotics. The peptide moiety is formed 

by hydroxypyridylhomothreonine, the nucleoside moiety consists of aminohexuronic acid 

with an N-glycosidically linked uracil or 4-formyl-4-imidazoline-2-one base. This work is 

intended to shed light onto the biosynthetic pathway that leads to the aminohexuronic acid 

moiety of nikkomycins. Commencing with the transfer of an enolpyruvyl moiety to the 3´position 

of UMP, which is catalyzed by NikO, a series of reactions leads to a rearrangement 

of the carbon skeleton and finally to the formation of aminohexuronic acid. The enzymes 

involved in these reactions are encoded on the nikIJKLMNO operon of S. tendae. The 

enzymes NikI, NikJ, NikK, NikL, NikM, and NikO were expressed heterologously in 

Escherichia coli, in order to characterize them biochemically and determine their role in the 

biosynthesis of nikkomycins. Furthermore, nikkomycin intermediates produced by 

Streptomyces mutants featuring disruptions of the respective genes should be analyzed, but no 

nikkomycins could be detected in their fermentation media. Comparisons of Nik enzyme 

sequences to those of known enzymes in the databases allow some predictions on the 

reactions each nik enzyme might catalyze. None of the enzymes was observed to convert 3´- 

EPUMP, the product of the NikO catalyzed reaction, so far. NikK is a pyridoxal-5-phosphate 

dependent aminotransferase, which is expected to introduce the amino group into the 

aminohexuronic acid moiety. Several amino acids were shown to serve as amino group 

donors, and L-glutamate was the most efficient one. NikJ possesses an iron-sulfur cluster and 

probably belongs to the family of radical SAM enzymes. Its role in nikkomycin biosynthesis 

is unclear, but it is expected to function as an oxidoreductase. Cleavage of Sadenosylmethionine 

could not be observed. Furthermore, NikS was expressed, an enzyme that 

is expected to play a role in the assembly of nikkomycins. 

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 P24189: “Bacterial bioluminescence” 

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” 

13

Invited Lectures 

1) A genomic and structural view of flavin-dependent proteins. International conference on 

cofactors, 14. July 2011, Turku, Finland 

2) From structure-based flavoenzymology to functional flavogenomics. Colloquium at the 

Max F. Perutz Laboratory - Vienna Biocentre, 27. October 2011, Vienna, Austria 

Publications 

1) Schrittwieser, J. H., Resch, V., Sattler, J., Lienhart, W.-D., Durchschein, K., Winkler, 

A., Gruber, K., Macheroux, P., and Kroutil, W.: Biocatalytic enantioselective oxidative 

C-C coupling by aerobic C-H activation of N-CH3 to yield novel berberine alkaloid 

derivatives, Angew. Chem. Int. Ed., 2011, 50:1068-1071. 

2) Schittmayer, M., Glieder, A., Uhl, M. K., Winkler, A., Zach, S., Schrittwieser, J., 

Kroutil, W., Macheroux, P., Gruber, K., Kambourakis, S., Rozzell, J. D., Winkler, M.: 

Old Yellow Enzyme catalyzed dehydrogenation of saturated ketones, Adv. Synth. 

Catal., 2011, 353:268-274. 

3) Durchschein, K., Fabian, W. M. F., Macheroux, P., Zangger, K., Trimmel, G., Faber, 

K.: Reductive biotransformation of nitroalkenes via nitroso-intermediates to oxazetes 

catalyzed by xenobiotic reductase A (XenA), Org. Biomol. Chem., 2011, 9:3364-3369. 

4) Gesell, A., Chavez, M. L. D., Kramell, R., Piotrowski, M., Macheroux, P., and Kutchan, 

T. M.: Heterologous expression of two FAD-dependent oxidases with (S)tetrahydroprotoberberine 

oxidase activity from Argemonas mexicana and Berberis 

wilsoniae in insect cells, Planta, 2011, 233:1185-1197. 

5) Macheroux, P., Kappes, B., and Ealick, S. E.: Flavogenomics: A genomic and structural 

view on flavin-dependent proteins, FEBS J., 2011, 278:2625-2634. 

6) Schrittwieser, J. H., Resch, V., Wallner, S., Lienhart, W.-D., Sattler, J. H., Resch, J., 

Macheroux, P., Kroutil, W.: Biocatalytic organic synthesis of optically pure (S)scoulerine 

and novel berbine and benzylisoquinoline alkaloids, J. Org. Chem., 2011, 

76:6703-6714. 

7) Schober, M., Gadler, P., Knaus, T., Kayer, H., Birner-Grünberger, R., Gülly, C., 

Macheroux, P., Wagner, U., Faber, K.: A stereoselective inverting sec-alkylsulfatase for 

the deracemisation of sec-alcohols, Org. Lett., 2011, 13:4296-4299. 

8) Resch, V., Schrittwieser, J. H., Wallner, S., Macheroux, P., Kroutil, W.: Biocatalytic 

oxidative carbon-carbon bond formation catalysed by berberine bridge enzyme: optimal 

reaction conditions, Adv. Synth. Catal., 2011, 353:2377-2383. 

9) Kappes, B., Tews, I., Binter, A., Macheroux, P.: PLP-dependent enzymes as potential 

drug targets for protozoan diseases, Biophys. Biochem. Acta, 2011, 1814:1567-1576. 

10) Binter, A., Oberdorfer, G., Hofzumahaus, S., Nerstheimer, S., Altenbacher, G., Gruber, 

K., Macheroux, P.: NikK is a pyridoxal 5’-phosphate dependent enzyme in nikkomycin 

biosynthesis, FEBS J., 2011, 278:4122-4135. 

11) Jankowitsch, F., Kühm, C., Kellner, R., Kalinowski, J., Pelzer, S., Macheroux, P., and 

Mack, M.: RosA is a novel N,N-8-amino-8-demethyl-D-riboflavin dimethyltransferase 

catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces 

davawensis, J. Biol. Chem., 2011, 286:38275-38285. 

14

Cell Biology Group 

Group leader: Günther Daum 

Postdoctoral Fellow: Karlheinz Grillitsch (ACIB) 

PhD students: Miroslava Spanova (till March 2011), Susanne Horvath, Martina Gsell, Vid V. 

Flis, Vasyl’ Ivashov, Lisa Klug, Claudia Schmidt, Barbara Koch, Birgit Ploier 

Master students: Brigitte Wagner, Gerald Mascher, Stefanie Horvath 

Technicians: Claudia Hrastnik (half time), Alma Ljubijankic (half time) 

Guest students: Giulia De Giglio (University of Torino, Italy); Suresh Nair (University of 

New South Wales, Sydney, Australia) 


Organelles divide the cell in different compartments which harbor distinct enzymatic steps 

and metabolic pathways. Therefore, organelles are the basis for regulated processes within a 

cell. All organelles are surrounded by membranes which do not only protect the interior from 

access but also govern communication within the cell. Our laboratory has a long standing 

tradition to study biogenesis and maintenance of biological membranes and assembly of lipids 

into organelle membranes using the yeast as an experimental model 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) biosynthesis of 

phosphatidylethanolamine (PE) in yeast organelles with emphasis on the role of the major PE 

synthesizing enzyme, the mitochondrial phosphatidylserine decarboxylase 1; (ii) non-polar 

lipid metabolism in the yeast with emphasis on lipid particle/droplet formation and 

mobilization of lipid depots by lipases and esterases; and (iii) isolation and characterization of 

organelle membranes from the yeast Pichia pastoris. 

Phosphatidylethanolamine, a key component of yeast organelle membranes 

Phosphatidylethanolamine (PE) is one of the major phospholipids of yeast membranes. It is 

highly important for membrane stability and integrity and thus also for cell function and 

proliferation. PE synthesis in the yeast is accomplished by four different pathways, namely 

(i) synthesis of phosphatidylserine (PS) in the endoplasmic reticulum and decarboxylation by 

mitochondrial phosphatidylserine decarboxylase 1 (Psd1p); (ii) synthesis of PS and 

conversion to PE by the Golgi localized Psd2p, (iii) the CDP-ethanolamine pathway 

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

route catalyzed by Ale1p and Tgl3p. To obtain more insight into biosynthesis, assembly and 

homeostasis of PE, single and multiple yeast mutants bearing defects in the respective 

pathways are required. 

15

Pathways of phosphatidylethanolamine formation in the yeast 

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, genetic screenings and DNA microarray analysis were 

performed which led to the discovery of a number of candidate genes. Grouping the 

respective gene products 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. The most promising candidates of this 

screening were investigated in some detail. Deletion mutants as well as overexpressing 

strains were used to analyze the influence of the novel gene products on lipid metabolism, 

membrane proliferation and cell growth. This study which is still in progress will contribute 

to our understanding of the complex network of phospholipid synthesis in the yeast. 

PE homeostasis in the yeast cell is linked to traffic of this phospholipid between various 

compartments. Currently, the link between PE metabolism and peroxisome proliferation is 

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

involved. Previous studies had suggested that PE formed through all four pathways (see 

above) in different subcellular membranes can be supplied to peroxisomes with comparable 

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

By means of various mutants and employing cell biological methods such as the use of 

permeabilized yeast cells PE transport to peroxisomes can be studied. The contribution of the 

different PE biosynthetic pathways to the supply of PE to peroxisomes and mechanisms of 

PE translocation between organelles are central aspects of this study. More recently, these 

investigations were extended to the supply of phosphatidylcholine (PC) to peroxisomes. Also 

in this case the emphasis is on the contribution of different PC forming pathways (PE 

methylation and CDP-choline pathway) and modes of PC translocation to this organelle. 

During the last year a major aspect of this project was characterization of the mitochondrial 

Psd1p with respect to 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 a most fruitful 

collaboration with the laboratory of Prof. N. Pfanner and colleagues, Freiburg, Germany, we 

investigated (i) the precise import route of Psd1p through mitochondrial membranes, (ii) the 

16

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. 

Assembly of Psd1p into mitochondrial membranes 

We also studied the link between PE metabolism and non-polar lipid storage. In the yeast 

Saccharomyces cerevisiae triacylglycerols (TG) are synthesized by the acyl-CoA dependent 

acyltransferases Dga1p, Are1p, Are2p and the acyl-CoA independent phospholipid: 

diacylglycerol acyltransferase (PDAT) Lro1p which uses PE as a preferred acyl donor. In this 

study we investigated a link between TG and PE metabolism by analyzing the contribution of 

the four different PE biosynthetic pathways (see above) to TG formation. In 

cki1∆dpl1∆eki1∆ mutants bearing defects in the CDP-ethanolamine pathway of PE synthesis 

both the cellular and the microsomal levels of PE were markedly decreased, whereas in other 

mutants of PE biosynthetic routes depletion of this aminoglycerophospholipid was less 

pronounced in microsomes. This observation is important because Lro1p similar to the 

enzymes of the CDP-ethanolamine pathway is a component of the ER. We concluded from 

these results that in cki1∆dpl1∆eki1∆ insufficient supply of PE to the PDAT Lro1p was a 

major reason for the strongly reduced TG level. Moreover, we found that Lro1p activity was 

lower in cki1∆dpl1∆eki1∆, although transcription of LRO1 was not affected. Our findings 

imply that TG and PE synthesis in the yeast are tightly linked, and TG formation by the 

PDAT Lro1p strongly depends on PE synthesis through the CDP-ethanolamine pathway. 

17

Storage of non-polar lipids in lipid droplets and mobilization 

Yeast cells like most other cell types have the capacity to store non-polar lipids. In the case of 

Saccharomyces cerevisiae triacylglycerols (TG) and steryl esters (SE) are the predominant 

lipid storage molecules which accumulate in subcellular structures named lipid 

particles/droplets. Upon requirement, TG and SE 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 formation and mobilization of TG and 

SE depots. 

Life cycle of yeast neutral lipids: Genes involved in non-polar lipid formation and 

degradation. 

Previous studies in our laboratory had identified Tgl3p, Tgl4p and Tgl5p as major yeast TG 

lipases. In addition, however, a number of candidate gene products with potential 

lipase/esterase activity were also identified. One of these polypeptides is Ldh1p, a lipid 

droplet associated hydrolytic enzyme. In collaboration with the laboratory of R. Erdmann, 

Bochum, Germany, we demonstrated that recombinant Ldh1p exerts esterase and TG lipase 

activities. The enzyme activity was abolished upon mutation of the conserved GXSXG-type 

lipase motif of the protein. Saccharomyces cerevisiae deleted of LDH1 was characterized by 

the appearance of giant lipid droplets and accumulation of non-polar lipids and phospholipids 

in LDs, indicative of a role of Ldh1p in maintaining lipid homeostasis. 

A major focus of our non-polar lipid project was the biochemical characterization of the three 

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

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

mobilization of TG, 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 TG fraction. 

Surprising evidence was obtained when the enzymology of the three TG lipases Tgl3p, Tgl4p 

18

and Tgl5p was studied. Motif search analysis indicated that Tgl3p, Tgl4p and Tgl5p do not 

only contain the TG lipase but also an acyltransferase motif. Indeed, all three enzymes exhibit 

lipase activity but also catalyze acylation of lysophosphatidylethanolamine and 

lysophosphatidic acid, respectively. Experiments using variants of lipases demonstrated that 

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

yeast lipases may play a dual role in lipid metabolism contributing to both anabolic and 

catabolic processes. This finding led us to investigate more deeply the biochemistry of yeast 

lipases. Currently, molecular properties of these enzymes including their topology on lipid 

droplets and the functional link between non-polar lipid formation and degradation are 

studied. 

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

major lipid droplet proteins from 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/droplets. Recently, a more precise 

yeast lipid droplet proteome analysis was initiated in collaboration with M. Karas, Frankfurt, 

Germany. This proteome study was combined with a lipidome investigation performed in 

collaboration with H. Köfeler, Medical University of Graz, Austria. In this study, we 

compared lipid droplet components from cells grown on glucose or oleate. This approach 

identified a number of lipid droplet 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 droplet proteome from cells grown on glucose or oleate. 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 

investigations of protein-lipid interaction on the surface of lipid droplets and provides 

fundamental evidence for the coordinated biosynthesis of lipid and protein components during 

biogenesis of this compartment. 

Identification of novel lipid droplet proteins from cell grown on glucose (YPD) or oleate 

(YPO). 

Another 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 TG and SE. In mutants lacking the ability to form lipid droplets, however, 

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

19

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 SE which form concentric shells in lipid droplets from wild type. In 

biological and artificial membranes fluorescence spectroscopy studies revealed that changes 

in membrane fluidity/rigidity are not result of absolute squalene levels, but are rather affected 

by the squalene to ergosterol ratio. In a fluid membrane environment squalene induces rigidity 

of the membrane, whereas in rigid membrane there is almost no additive effect of squalene. 

Although squalene is not a typical membrane lipid it may be regarded as a mild modulator of 

biophysical membrane properties. 

Pichia pastoris organelles and lipids 

Doctoral Thesis completed 

Miroslava Spanova: Neutral Lipid Storage in Yeast 

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 focused on the 

biochemical characterization of the plasma membrane and 

secretory organelles from Pichia pastoris. Moreover, lipid 

droplets from Pichia pastoris were isolated and analyzed 

with respect to their lipids and proteins. Mutants bearing 

defects in non-polar lipid formation of Pichia pastoris were 

constructed. Detailed investigations with these strains are 

currently in progress. Methods of Pichia pastoris organelle 

characterization include standardized techniques of lipidome 

and proteome analyses. 

Transmission electron micrographs of Pichia pastoris wild 

type cells grown on glucose (A), glycerol (B), and methanol 

(C). Pictures shown be courtesy of Dr. G. Zellnig, University 

of Graz, Austria 

Squalene, a natural triterpene, is a key intermediate in ergosterol synthesis of the yeast and 

present in the cell only at minor amounts under standard conditions. For the present study we 

constructed strains of Saccharomyces cerevisiae accumulating squalene to perform 

fundamental research but also as a possible source for biotechnological production of this 

lipid. We first addressed localization of squalene within the cell and its possible lipotoxic 

effect in the yeast. We showed that the highly hydrophobic squalene preferentially localizes to 

lipid particles/droplets. In this compartment it decreases the order which is created by steryl 

esters forming concentric layers around a core of triacylglycerols. We also observed that 

squalene did not exhibit a lipotoxic effect even in mutant strains which are unable to form 

lipid particles. In strains devoid of lipid particles large amounts of squalene were stored in 

20

membranes. This finding was surprising because squalene is not a typical membrane lipid and 

cannot actively contribute to membrane formation per se due to its hydrophobic nature. This 

observation led us to investigate the influence of squalene on membrane stability. We showed 

that endoplasmic reticulum membranes became more rigid when enriched with squalene, 

whereas plasma membrane samples became softer. The latter finding was in line with 

increased sensitivity of cells to high and low pH, high salt or detergent concentration. 

However, the combination of squalene with low or high amounts of sterols in a membrane 

seems to be important for the squalene effect. In summary, 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. 

Master Theses completed 

Brigitte Wagner: Cloning and characterization of novel hydrolases in Saccharomyces 

cerevisiae 

This Master Thesis was focused on the search of novel hydrolases in the yeast Saccharomyces 

cerevisiae. Previous studies provided evidence for several candidate enzymes of this type. 

Thus, the aim of this work was to identify and to characterize some of them. It was 

hypothesized that the enzymes investigated might also be involved in neutral lipid metabolism 

of S. cerevisiae. During this investigation efforts were focused on the gene YBR056w which 

was successfully cloned into the E. coli expression vector pET21b. Hydrophobicity programs 

predicted no transmembrane regions for YBR056wp and proposed rather a globular protein 

without any signal peptides. The recombinant protein was overexpressed and purified via a 6x 

Histidin-tag. Finally, enzymatic analysis was performed. Enzyme activity measurements 

showed that YBR056wp indeed possesses hydrolytic activity with a vmax of 0.18 

µmol/min/mg and a Km of 5.07 mM with p-nitrophenol-butyrate as substrate. The addition of 

different ions did not improve the enzyme activity. Furthermore, the pH optimum was 

determined to be pH 6.0, which lies in the normal range of most enzymes. Moreover, the 

disturbing effect of detergents on enzyme activity was shown. An influence of YBR056wp on 

neutral lipid metabolism of the yeast could not be demonstrated. Recently, the protein Ldh1p 

was identified as a novel yeast hydrolase by Debelyy et al. (Eukaryot. Cell, 10, 776–781, 

2011). Thus, Ldh1p served as a positive control for hydrolase activity measurements. For this 

purpose, also Ldh1p was overexpressed and purified. The maximal reaction rate of 0.36 

µmol/min/mg as well as the Km value of 0.89 mM correlated reasonably with the published 

data. Further characterization of this enzyme showed that Ldh1p has a pH optimum of pH 7.2 

and detergents had a harmful effect on enzyme activity. Finally, it was shown that ions had no 

positive effect on enzyme activity. In summary, YBR056wp and Ldh1p are two novel 

hydrolases of the yeast S. cerevisiae with similar properties. 

Gerald Mascher: Gph1p, Rsb1p and Gpm2p: investigation of their connection to 

phospholipid metabolism in the yeast Saccharomyces cerevisiae 

Phosphatidylethanolamine (PE) is one of the major phospholipids in membranes of the yeast 

Saccharomyces cerevisiae. Its synthesis is accomplished by a network of reactions which 

comprises four different pathways. Psd1p (phosphatidylserine decarboxylase 1) which 

contributes most to PE formation in yeast is localized to the inner mitochondrial membrane. 

21

To study the general role of PE in yeast and effects of an unbalanced PE level, genome wide 

effects of a ∆psd1 deletion were analyzed in a DNA microarray study. In the deletion mutant 

54 genes were up-regulated compared to wild type. Three of these genes, GPH1, RSB1 and 

GPM2 were analyzed in more detail during the present study. To study possible physiological 

functions of the gene products, yeast strains lacking and overexpressing the respective genes 

were investigated for growth phenotype on different carbon sources and phospholipid 

patterns. Additionally, the localization of the GFP-tagged gene products was studied by 

fluorescence microscopy, and the role of Gph1p on cell structure was analyzed by electron 

microscopy. Furthermore, the influence of the genetic wild type background on the 

phospholipid composition was part of this study. GPH1 which encodes a glycogen 

phosphorylase seems to be essential for osmotic stability, resistance to SDS, formation of 

lipid particles and a balanced phospholipid metabolism. The predicted role in osmotic stability 

and resistance to SDS is linked to the genetic background. The two wild type strains BY4741 

and BY4742 resulted in a different phospholipid composition of the plasma membrane. The 

higher PE level of BY4741 combined with a GPH1 deletion caused osmotic instability and 

strong sensitivity to SDS. Lipid analyses showed that Gph1p may be involved in synthesis 

and transport of phosphatidylcholine (PC) and play also a role in neutral lipid storage. All 

these findings confirmed that Gph1p is a global player in lipid metabolism, but its specific 

function remained unclear. The second gene of interest, RSB1, does not play a special role in 

phospholipid metabolism. Fluorescence microscopic analysis showed that Rsb1p is enriched 

in PAM (plasma membrane associated membranes of the ER) where it may be involved in 

LCB (sphingoid long chain base) transport and topology in bilayer membranes. These 

functions seem to be important for maintaining membrane integrity, for example during 

depletion of PE. A connection of GPM2 to lipid metabolism was not observed. Gpm2p is 

predicted to be a non-functional homologue of Gpm1p which acts as phosphoglycerate 

mutase during glycolysis and gluconeogenesis. 


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

Germany 

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

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

Frankfurt, Germany 

C. F. Clarke, Department of Chemistry and Biochemistry, University of California, Los 

Angeles, CA, USA 

S. Moye-Rowley, Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, 

USA 

Research projects 

FWF P21429: Phosphatidylserine decarboxylase 

FWF P23029 Lipases of the yeast Saccharomyces cerevisiae 

FWF TRP 009 Pichia Lipidomics (Translational Research) 

FWF PhD Program: Molecular Enzymology 

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

Production 


22

1. G. Daum (Guest lecturer) 

University Rovira i Virgili Campus Sescelades Marcel, Tarragona, Spain 

Lecture Series: Yeast Lipids (Master Program). 14-18 February 2011 

2. G. Daum 

Central Institute of Medicinal and Aromatic Plants, Lucknow, India 

Yeast as a model system for genetics, biochemistry and cell biology from the viewpoint 

of a lipidologist. 14 March 2011 

3. G. Daum 

Central Institute of Medicinal and Aromatic Plants, Lucknow, India 

Cell biology of lipid storage and mobilization in the yeast. 15 March 2011 

4. G. Daum 

Council of Scientific and Industrial Research, Jawaharla Nehru University, Delhi, India 

Cell biology of lipid storage and mobilization in the yeast. 25 March 2011 

5. M. Spanova, D. Zweytick, K. Lohner, E. Leitner, A. Hermetter and G. Daum 

10 th Yeast Lipid Conference, Oulu, Finland, 26-29 May 2011 

Squalene - another storage lipid of the yeast. 

6. G. Daum (Normann Medal Award Lecture) 

9 th Euro Fed Lipid Congress, Oils, Fats and Lipids for a Healthy and Sustainable World, 

Rotterdam, The Netherlands, 18-21 September 2011 

Lipid Storage: Yeast we can! 

7. G. Daum 

Amyris, Lipidfest Symposium, Emeryville, CA, USA 

Lipid Storage: Yeast we can! 11 October 2011 

8. G. Daum 

Department of Molecular and Cell Biology, U. C. Berkeley, CA, USA 

Lipids of the yeast. 13 October 2011 

Publications 

1. Grillitsch, K. and Daum, G. 

Triacylglycerol lipases of the yeast 

Front. Biol. 6 (2011) 219-230 

2. Debelyy, M. O., Thoms, S., Connerth, M., Daum, G. and Erdmann, R. 

Involvement of the yeast hydrolase Ldh1p in lipid homeostasis. 

Eukaryot. Cell 10 (2011) 776-781 

3. Thoms, S., Debelyy, M. O., Connerth, M., Daum, G. and Erdmann, R. 

The putative yeast hydrolase Ldh1p is localized to lipid droplets. 

Eukaryot. Cell 10 (2011) 770-775 

4. Grillitsch, K., Connerth, M., Köfeler, H., Arrey, T. N., Rietschel, B., Wagner, B., Karas, 

M. and Daum, G.: 

Lipid particles/droplets of the yeast Saccharomyces cerevisiae revisited: Lipidome 

meets Proteome. 

Biochim. Biophys. Acta 1811 (2011) 1165-1176 

5. Horvath, S. E., Wagner, A., Steyrer, E. and Daum, G. 

Metabolic link between phosphatidylethanolamine and triacylglycerol metabolism in the 

yeast Saccharomyces cerevisiae. 


23

6. Spanova, M. and Daum, G. 

Squalene – Biochemistry, Molecular Biology, Process Biotechnology and Applications 

Eur. J. Lipid Sci. Technol. 113 (2011) 1299-1320 

7. Athenstaedt, K. and Daum, G. 

Lipid storage: Yeast we can! 


Awards 

Karlheinz Grillitsch 

Award for best poster 

Characterization of Pichia pastoris plasma membrane 

Non-conventional yeasts in the Postgenomic Era (NCY-2011), Lviv, Ukraine, 11-14 

September 2011 

Günther Daum 

Normann Medal of the German Society of Lipid Research (DGF) 

Award Lecture: Lipid Storage: Yeast we can! 

9 th Euro Fed Lipid Congress, Oils, Fats and Lipids for a Healthy and Sustainable 

World, Rotterdam, The Netherlands, 18-21 September 2011 

Susanne Horvath spent a research semester at the Institute of Biochemistry and Molecular 

Biology, ZBMZ, University of Freiburg, Germany, in the laboratory of N. Pfanner. This 

stay abroad was supported by the FWF PhD Program Molecular Enzymology. 

24

Molecular Biology Group 

Group leader: Karin Athenstaedt 

Two triacylglycerol synthases of 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 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. Despite its industrial application information about TAG (lipid) 

metabolism in Yarrowia lipolytica is rather limited. Homology searches with TAG synthases 

of other yeast species as queries highlighted two candidate gene-products of the oleaginous 

yeast potentially catalyzing the formation of TAG. In the respective single deletion mutants 

the amount of TAG is significantly decreased. 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. Heterologous expression of both polypeptides restored the formation of 

lipid particles in host cells. Lipid analyses revealed that the neutral lipid core of lipid particles 

in the respective host cells was formed of TAG (Fig. 1). 

A 

B 

S 

STE 

E 

S + STE 

TAG 

E 

1 2 3 4 5 

Biosynthesis of phosphatidic acid in yeast 

Figure 1: Heterologous expression of Yarrowia 

lipolytica TAG synthases restores the formation of 

TAG (lanes 4 and 5) in mutant cells of the budding 

yeast Saccharomyces cerevisiae otherwise lacking 

neutral lipids (lane 3). Lane 1: standard containing 

ergosterol (E), triacylglycerols (TAG) and sterol esters 

(STE); lane 2: neutral lipid pattern of the 

corresponding Saccharomyces cerevisiae wild-type 

strain. S: squalene. 

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 

25

and Gat2p, and two major 1-acyl glycerol-3-phosphate acyltransferases, Slc1p and Ale1p. 

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. 

Localization studies revealed that Gat1p is associated with lipid particles and the endoplasmic 

reticulum, whereas Gat2p is exclusively localized to the latter compartment in wild-type 

background (Fig. 2). 

A 

B 

LP 

Most interestingly, the distribution pattern of both glycerol-3-phosphate acyltransferases 

changes in the absence of the respective counterpart. Gene expression analyses revealed that 

several genes conversely respond to the presence or absence of GAT1 and GAT2 indicating 

that phosphatidic acid formed by Gat1p or Gat2p has different functions. These indicated 

differences in metabolism of phosphatidic acid species as well as potential interactions of 

GAT-proteins with candidate proteins are currently under investigation. 

International Cooperations 

V. Zaremberg, Department of Biological Sciences, University of Calgary, Canada 

J. Goodman, Department of Pharmacology, University of Texas Southwestern Medical 

School, USA 

Research project 

FWF P21251: Biosynthesis of phosphatidic acid in yeast 

Publications 

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 

arrow heads). In contrast, GFP-Gat2p is only 

present in the endoplasmic reticulum (B). 

1) Athenstaedt, K. 

YALI0E32769g (DGA1) and YALI0E16797g (LRO1) encode major triacylglycerol 

synthases of the oleaginous yeast Yarrowia lipolytica. 


2) Athenstaedt, K. and Daum, G. 

Lipid storage: Yeast we can! 


26

Biophysical Chemistry Group 

Group leader: Albin Hermetter 

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

Halasiddappa, Bojana Stojcic, Franziska Vogl 

Master students: Gabriel Pürstinger, Hannah Jaritz, Luise Britz 

Technician: 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 supramolecular systems. 

Section 1 of the following report summarizes our studies on the toxic effects of oxidized 

phospholipids on the cells of the vascular wall and on cancer cells with emphasis on ceramide 

as a lipid mediator of lipid toxicity. 

Section 2 describes the development of fluorescence methods for functional proteomic 

analysis of lipolytic enzymes in animal and human cells. 

1. Oxidized phospholipids and disease – Lipid toxicity 

Polyunsaturated phospholipids in cell membranes and plasma lipoproteins are modified under 

the conditions of oxidative stress. Oxidized phospholipids (OxPL) are formed, showing a 

great variety of biological activities. Many of these compounds are toxic in cells. On the one 

hand, lipid toxicity may lead to pathophysiological consequences. In the cells of the arterial 

wall, OxPL are involved in the initiation and progress of atherosclerosis. On the other hand, 

these compounds might elicit beneficial effects. We recently found that some OxPL 

preferably kill tumor cells and therefore could possess therapeutic potential in cancer 

treatment (patent application filed). 

1.1. Toxicity of oxidized phospholipids in vascular cells 

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 ESF project 

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 position sn-1 and short 

27

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 

sphingolipidome, the enzyme activities responsible for ceramide homeostasis, the proteome 

and the transcriptome to find the primary molecular targets and their downstream elements 

that are the functional components of lipid-induced cell death. 

Sphingomyelin 

acid 

Sphingomyelinase 

Ceramide 

JNK 

p38 MAPK 

Caspase 3 

Oxidized - 

O 

Phospholipids 

H 

O 

O 

O 

O 

O 

O 

O P 

O 

O 

OH 

+ 

N 

O 

O 

O 

O 

O P 

O 

OH 

N + 

O 

ERK 

AKT/PKB 

NFkB 

PROLIFERATION 

SURVIVAL 

OXPHOS-EuroMEMBRANE 

1.2. Toxicity of oxidized phospholipids in cancer cells 

Oxidized phospholipids generate apoptotic 

blebs in melanoma cells 

Cancer is a complex disease characterized by genetic 

mutations that lead to uncontrolled cell growth and 

spread of abnormal cells. One in every three cancers 

diagnosed is skin cancer, which is skin growth of 

melanocytic and non-melanocytic cells with 

differing causes and varying degrees of malignancy. 

Melanomas which derive from pigment-producing 

melanocytes in the basal layer of the epidermis 

represent the most dangerous form of skin cancer 

and although not being the most common skin 

cancer type, they are responsible for most skin 

cancer deaths. These tumors have a high chance of 

metastasizing and becoming lethal. Surgical removal 

of the tumor is only possible at a very early stage, 

and chemotherapy as an overall strategy is not very 

effective in treating melanomas. Only 15 % to 20 % of patients respond to chemotherapy 

which typically works for less than a year and has little or no effect on survival time. In 

28

collaboration with Dr. Schaider from the Department of Dermatology at Medical University 

Graz, we found that oxidized phospholipids induce apoptosis in various types of skin cancer 

cells, including melanomas and non-melanoma skin cancer. To understand the basis of lipidinduced 

cell death, we studied the uptake and stability of these lipids in different skin cancer 

cell lines. In addition, we analyzed apoptotic signaling pathways associated with sphingolipid 

metabolism and screened for potential protein and membrane lipid targets. In summary, the 

toxic lipid effects were much more pronounced in cancer cells, especially on metastatic 

melanoma cells, than in healthy cells. Therefore, the results of this study can be considered a 

useful basis of a new generalized approach for the therapy of skin cancer, which allows 

bypassing the genetic heterogeneity of tumor cells. 

European patent application filed by TU Graz. 

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. 

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 

Inactive 

Active 

Inactive 

Inactive 

Inactive 

Inhibited 

BG 

BG 

RG 

RG 

NBD 

NBD 

RG: 

R e a c t i v e p h o s p h o n a t e g r o u p , 

irreversibly inhibits the catalytic serine 

BG: 

Binding group: is specifically recognized 

by the active enzyme 

NBD : fluorescent tag l : 488 nm; l : 540 nm 

29 

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 

30 

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 

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

background, environment, metabolic state and disease. 

Doctoral Thesis completed: 

Ute Stemmer: Toxicity, uptake and targeting of oxidized phospholipids in cultured 

macrophages. 

The uptake of oxidized low-density lipoprotein (oxLDL) by the macrophages of the arterial 

wall and the accumulation of apoptotic cells in atherosclerotic lesions are hallmarks of 

atherosclerosis. The harmful effects of oxLDL are largely mediated by the intrinsic toxicity of 

a great variety of lipid oxidation products that are generated in LDL under the conditions of 

oxidative stress. These compounds comprise oxidized sterols, oxidized fatty acid derivatives 

and oxidized glycerophospholipids. Oxidized phospholipids may contain a modified longchain 

carboxylic acid or a truncated acyl residue with a polar functional group at its ω-end. 

Typical derivatives of truncated phospholipids are the carboxy-phospholipid 1-palmitoyl-2glutaroyl-sn-glycero-3-phosphocholine 

(PGPC) and the chemically reactive aldehydophosphospholipid 

1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 

which are oxidation products of arachidonoyl-phosphatidylcholine. The latter lipid can 

undergo Schiff base formation with amino groups of proteins, thereby modulating their 

properties and functions. It was the aim of the first part of this doctoral thesis to find out

whether and to what extent the biological effects of oxLDL are mediated by truncated 

diacylphospholipids in cultured macrophages. For this purpose, we used chemically defined 

PGPC and POVPC as well as their natural 1-O-hexadecyl analogs and determined their 

toxicity in the macrophage-like cell line RAW 264.7 and bone marrow-derived macrophages 

(BMM). PGPC and POVPC induced apoptosis in both cell types, the bone marrow-derived 

cells being slightly more susceptible to phospholipid toxicity than the RAW 264.7 cells. 

POVPC rapidly activated acid sphingomyelinase (aSMase) in cultured RAW 264.7 

macrophages. If the expression of aSMase was abolished by a specific inhibitor, the cells 

became more resistant to POVPC-induced apoptosis, showing that the activity of this enzyme 

is causally linked to POVPC toxicity in macrophages. The second part of this doctoral thesis 

was devoted to the cellular uptake and the primary molecular targets of the oxidized 

phosphatidylcholines. For this purpose, we used the fluorescently-labeled analogs of PGPC 

and POVPC for visualization of lipids in live cells and lipid-protein complexes in acrylamide 

or agarose gels. The fluorescent analogs of PGPC and POVPC were easily taken up by the 

RAW 264.7 macrophages irrespective of the lipid presentation. Whereas POVPC was initially 

scavenged by covalent reaction with the components of the plasma membrane, PGPC was 

quickly internalized. Despite the covalent binding to its lipid and protein targets, POVPC is 

freely exchangeable between membranes and (lipo-) protein surfaces. As a consequence, this 

lipid represents a toxic compound which is active not only at the site of its formation but also 

in cells far distant from areas of oxidative stress. We isolated and identified the protein targets 

in cultured RAW 264.7 macrophages, which formed covalent Schiff base adducts with BY- 

POVPE. The respective polypeptides are involved in membrane transport, stress response, 

apoptosis and lipid metabolism. The identification of a considerable number of potential 

POVPC protein targets supports the assumption that POVPC interacts with multiple sites and 

not only with the traditional specific receptors (U. Stemmer et al., 2 manuscripts in revision). 

International cooperations: 

T. Futerman, Department of Biological Chemistry, Weizman Institute, Rehovot, Israel 

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 

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 

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

Cornell University, Ithaca, USA 

R.P. Kühnlein, Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für 

Biophysikalische Chemie, Göttingen, Germany. 

Research projects: 

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

Phospholipases (GOLD 3 project-Genomics of Lipid-associated Disorders, KFU Graz). 

FWF Special Research Program (SFB) LIPOTOX (KFU Graz): Toxicity of oxidized 

phospholipids in macrophages (until March 2011). 

31

FWF Doctoral Program Molecular Enzymology: Enzymes of ceramide signaling in response 

to oxidized phospholipids. 

FWF, ESF-EuroMEMBRANE-OXPHOS: Protein targets and apoptotic signaling of oxidized 

phospholipids 

Invited lecture: 

Toxicity of oxidized phospholipids in macrophages. Effects on ceramide synthesis. 

COST Workshop CM0603 Free Radicals in Chemical Biology, June 14th – 17th, 2011 

Ruđer Boškovic Institute , Zagreb, Croatia 

Publications: 

1. Watschinger K, Fuchs JE, Yarov-Yarovoy V, Keller MA, Golderer G, Hermetter A, 

Werner-Felmayer G, Hulo N, Werner ER. Catalytic residues and a predicted structure of 

tetrahydrobiopterin-dependent alkylglycerol monooxygenase. Biochem J, Epub ahead of 

print, 2011. 

2. Birner-Gruenberger R, Lange J, Bickmeyer I, Hermetter A, Kollroser M, Rechberger 

GN, Kühnlein RP. Functional fat body proteomics and gene targeting reveal in vivo 

functions of drosophila melanogaster α-Esterase-7. Insect Biochem & Mol Biol, Epub 

ahead of print, 2011. 

3. Stemmer U, Ramprecht C, Zenzmaier E, Stojcic B, Rechberger G, Kollroser M, 

Hermetter A. 

Uptake and protein targeting of fluorescent oxidized phospholipids in cultured RAW 

264.7 macrophages. 

Biochim. Biophys .Acta, in press (2011) 

European patent application : 

Phospholipid compounds for use in cancer treatment. 

Inventors: A. Hermetter, C. Ramprecht, H. Schaider 

32

Group Chemistry of Functional Foods 

Group leader: Michael Murkovic 

PhD students: Yuliana Reni Swasti 

Diploma students: Vanessa Verient, Wolfgang Sindler, Evelina Dimitrova 

International students: Monika Ugintaite, Ausra Degutyte, György Gyana, Tarek Gamal 

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; Zuzana Ciesarova, Food Research 

Institute Bratislava, Slovakia 


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. 

Polymerization of furfuryl alcohol during roasting of coffee 

Analysis of furfuryl alcohol oligomers from a model system and from roasted coffee using 

Ion Trap MS: The dimeric and trimeric furfuryl alcohol were also analyzed with Ion Trap MS. 

From MS1 results showed that the abundant ion is m/z 161 and m/z 241 which refer to 

dimeric and trimeric furfuryl, respectively. Those 2 abundant ions were then fragmented and 

showed the same way of fragmentation as in LC/MS analysis as can be seen in its MS2 and 

MS3. The dimeric furfuryl alcohol also found roasted coffee that was roasted at 210 °C for 4 

minutes using a household roasting machine. 

33

Figure 1. MS2 of dimeric of furfuryl alcohol in ion tap analysis. 

Green coffee does not contain any furfuryl alcohol. However, furfuryl alcohol is formed 

during roasting. After 3 min of roasting at 210 °C the formation of furfuryl alcohol starts with 

a significant increase during continued roasting and reduced at 5 min roasting. This means 

that a degradation of the 1,2-enediol (key intermediate in the isomerization reaction of 

fructose and glucose) and the degradation of quinic acid could occur. Furfuryl alcohol 

oligomers are also found after 3 min of roasting and are also reduced after 5 min of roasting. 

The oligomers that were identified with LC-MS is the dimer and acid condition also occur 

during coffee roasting. The dimer is present because during degradation a 1,2-enediol also 

form acid condition by producing formic acid. Therefore, furfuryl alcohol is able to 

polymerize under that conditions and contribute in the formation of brown color. 

Master Thesis completed 

Tajana Golukova: Formation of potentially toxic furan derivatives in foods 

During processing and especially roasting of food, many substances are formed by the 

Maillard reaction and caramelization. Many of the formed substances contribute to the flavor, 

but some of them can be harmful like HMF (5-hydroxymethylfurfural), HMFA (5hydroxymethyl-2-furoic 

acid), furfural and FA (furfuryl alcohol). In the present work HPLC 

methods for the determination of these substances were established, optimized, and afterwards 

adapted to the analyses of food. Coffee is well known for its high content of Maillard 

Reaction Products (MRPs) and also for the high content of furan derivatives. Therefore a 

focus was laid on coffee and the development of HMF, furfural and FA during the roasting 

process at 210 °C. Another aim was to find the precursors of HMFA in coffee with the 

purpose to explain the high concentration in roasted coffee. Furfural was derivatized with 

DNPH (2,4-dinitrophenylhydrazine) to get a better selectivity. Furfural was only found in 

small quantities in filter coffee (6,36 µg/g) and balsamic vinegar (below the LQ). High 

contents of furfuryl alcohol were observed in filter coffee (1430 µg/g) and instant coffee (267 

µg/g). Furthermore a low concentration was found in pineapple juice (3,28 µg/g). HMF was 

detected in plum juice (5,98 µg/g), coffee substitute (38,4 µg/g), balsamic vinegar (59,1 

µg/mL), caramel-syrup (33,9 µg/mL), filter coffee (187 µg/g) and instant coffee (869 µg/g). 

The roasting profile shows that the maximum concentrations of HMF (152 µg/g) and furfural 

(3,51 µg/g) occur after 2 minutes. In contrast the highest concentration of furfuryl alcohol 

34

(1340 µg/g) was found after 5 minutes of roasting. To find an explanation of the high HMFAcontent 

in coffee, several model systems were developed to simulate the processes of the 

Maillard reaction during the roasting of coffee. Following model systems were investigated: 

1. Glyceraldehyde + Na-pyruvate 2. Alanine + glyceraldehyde 3. Alanine + sucrose 4. Pectin 

5. Galacturonic acid 6. Glucuronic acid 7. Glucono-delta-lactone Only glucono-�-lactone and 

glyceraldehyde with Na-pyruvate generate HMFA. However, the HMFA concentrations were 

too low to explain the excessive formation in coffee. 


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

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

H.R. Glatt, Deutsches Institut 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, 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. Thongraung, Prince of Songkla University, Hatyai, Thailand 

D.W. Marseno, Gadjah Mada University 

Research project 

European Network of Excellence: EuroFIR European Food Information Resource 

Global Network of University Chairs on Innovation (UNCHAIN) 

Mitigation of acrylamide in bread by application of asparaginase 


1) Murkovic, M.: 

Pumpkin Seed Oil. - in: Institutes Seminar. Wuhan 


Fat oxidation and antioxidant. - in: Institutes Seminar. University of Gadjah Mada, 

Yogyakarta 


Antioxidants - possible implications on human health. - in: Institutes Seminar. 

Guangzhou 


Fat oxidation and antioxidants. - in: Institutes Seminar. University of Palembang 


Formation of carcinogenic substances during heating of foods. - in: Institutes Seminar. 

University of Palembang 


Fat oxidation and antioxidants. - in: Institutes Seminar. University of Mataram 

35


Formation of Carcinogens during Cooking. - in: Pharma and Food Lecture Series. Wien 


Heat induced carcinogenic compounds in foods. - in: Recent Advances in Food 

Research. Bratislava 


How antioxidants could contribute to a healthier food. - in: With Food to Health. Tuzla 


Formation of carcinogenic substances during heating of foods. - in: 2nd International 

Congress: Engineering, Ecology and Materials in the Processing Industry. Jahorina 

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

Mass spectrometric characterization of triglyceride oxidation. - in: 2nd MS Food Day. 

Trieste 

Publications 

1. Dimitrovska, M.; Bocevska, M.; Dimitrovski, D.; Murkovic, M. 

Anthocyanin composition of Vranec, Cabernet Sauvignon, Merlot and Pinot Noir 

grapes as indicator of their varietal differentiation. European Food Research and 

Technology 232 (2011) 591 - 600 

2. Zeb, A.; Murkovic, M. 

Carotenoids and Triacylglycerols Interactions during Thermal Oxidation of Refined 

Olive oil. Food Chemistry 127 (2011) 1584 – 1593 

3. Glatt, H.; Schneider, H.; Murkovic, M.; Monien, B. H.; Meinl, W. 

Hydroxymethyl-substituted furans: mutagenicity in Salmonella typhimurium strains 

engineered for expression of various human and rodent sulfotransferases. Mutagenesis 

(2011) in press 

36

Lectures and Laboratory Courses 

Aktuelle Trends und Ergebnisse in der 

Analytischen Chemie und Lebensmittelchemie 

2 VO Günther K 

Anleitung zu wissenschaftlichen Arbeiten aus 2 SE Daum G, Hermetter A, 

Biochemie und Molekularer Biomedizin 

Macheroux P 

Anleitung zu wissenschaftlichen Arbeiten aus 2 SE Daum G, Hermetter A, 

Biochemie und Molekularer Biomedizin 

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 

bei der Verarbeitung 

2 SE Murkovic M 

DissertantInnenseminar 1 1 SE Faculty 

DissertantInnenseminar 2 1 SE Faculty 

Enzymatische und mikrobielle Verfahren in der 

Lebensmittelherstellung 

2 VO Murkovic M 

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 

37

Molekulare Gastronomie und 

Lebensmittelverarbeitung II 

1 EV Murkovic M 

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 9 PR Macheroux P, Daum G, 

Biomedizin 1 

Hermetter A, Murkovic M, 

Waldner-Scott I 

Projektlabor Biochemie und Molekulare 9 PR Macheroux P, Daum G, 

Biomedizin 2 

Hermetter A, Murkovic M, 

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 

Spezielle Kapitel der Lebensmittelchem.- u. 

technologie I 


Spezielle Kapitel der Lebensmittelchemie- und 

techn.II 


Strukturanalyse in Biophysik u. 

Materialforschung 

2 VO Laggner P 

Unterrichtspraxis 2 SE Macheroux P 

Unterrichtspraxis 2 SE Macheroux P 

Wissenschaftliches Kolloquium für 

DissertantInnen 1 

1 SE Faculty 

Wissenschaftliches Kolloquium für 

DissertantInnen 2 

1 SE Faculty 

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

Staff Members of the Institute of Biochemistry, TU - Institut für ...

Create successful ePaper yourself

Delete template?

Save as template?