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. Ines Waldner-Scott
(ines.waldner-scott@tugraz.at; Tel.:+43-(0)316-873-6454)
DI Tanja Knaus
(tanja.knaus@tugraz.at; Tel.:+43-(0)316-873-6463)
DI Alexandra Binter
(alexandra.binter@tugraz.at; Tel.: +43-(0)316-873-6453)
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
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Brief History of the Institute of Biochemistry
The Institute of Biochemistry and Food Chemistry was born out of the division of the Institute
of Biochemical Technology, Food Chemistry and Microchemistry of the former School of
Technology Graz. Together with all the other chemistry institutes, it was located in the old
Chemistry Building on Baron Mandell's ground, corner Technikerstrasse-Mandellstrasse.
1929 The Institute of Technical Biochemistry and Microbiology moved to the building of
the Fürstlich-Dietrichstein-Stiftung, Schlögelgasse 9, in which all the biosciences were
then concentrated.
1945 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
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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.
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Highlights of 2009
In April 2009 the restructuring of the Faculty of Technical Chemistry, Chemical Engineering
and Biotechnology resulted in a partial reunification of the Institute of Food Chemistry and
Technology with the Institute of Biochemistry. During this reshaping Michael Murkovic with
his research group "Chemistry of Functional Foods” joined the Institute of Biochemistry.
The research group of Michael Murkovic is mainly interested in
the evaluation of reaction mechanisms in foods with respect to the
reactivity of antioxidants in relation to the formation of
carcinogenic substance during heating of foods. Merging of the
two research units has led to a reinforcement of the infrastructure
with emphasis on techniques of liquid chromatography and the
identification of small molecules.
As in the years before, the group of Peter Macheroux was very active in publishing their
research results in scientific journals. Among the published articles, one was again selected as
best paper of the week by the editors of the Journal of Biological Chemistry (“Hold on to that
flavin” published in July). This publication also marked the end of a very successful PhD
thesis project submitted by Andreas Winkler who left the institute and joined the Max-Planck-
Institute of Medical Research in Heidelberg, Germany, as an EMBO long-term postdoctoral
fellow. Just before Christmas, Peter Macheroux was awarded the Styrian Research Award for
studies on redox-regulated degradation of proteins by the quinone reductase-proteasome
complex. This project dates back to Peter Macheroux’ early days at the institute and was
supported by several outstanding students and postdocs, most notably Sonja Sollner, Andrea
Wagner, Sigrid Deller and Markus Schober.
P. Macheroux (right), Styrian Research Award 2009
4
G. Daum (right), Euro Fed Lipid Conference 2009
As in previous years, research in the laboratory of Günther Daum was supported by grants of
the Austrian Science Fund (FWF). In 2009, a new project devoted to “Phosphatidylserine
Decarboxylases of the Yeast” started with two new graduate students, Martina Gsell and Vid
V. Flis. In 2009, another project named ACIB (Austrian Center of Industrial Biotechnology)
supported by the Austrian Research Promotion Agency (FFG) and industry was approved. In
this joint project Günther Daum’s group will be involved in applied research devoted to
Pichia pastoris cell biology. Another highlight of 2009 was the Euro Fed Lipid Conference
(European Federation for the Science and Technology of Lipids) which was held in October

2009 in Graz under the chairmanship of Günther Daum. This conference with more than 600
participants was not only an interesting meeting which combined fundamental and applied
research but also an important event for the lipid community in Graz. Günther Daum will also
chair a FEBS workshop devoted to Microbial Lipids which will take place in May 2010 in
Vienna, Austria.
Basic research in Albin Hermetter’s group was performed in three projects focusing on lipid
(patho)physiology. His contribution to the GOLD project (speaker R. Zechner, IMB,
University of Graz, funded by the GEN-AU program of the BM.W_F) is devoted to the
functional proteomic analysis of (phospho)lipases relevant to lipid-associated disorders. In the
SFB LIPOTOX (FWF project, speaker R. Zechner) and the recently approved
EuroMEMBRANE consortium OXPHOS (speaker P. Kinnunen, University of Helsinki), A.
Hermetter studies the toxicity of oxidized phospholipids in the cells of the arterial wall
(funded by FWF). Research in this field is also supported by the PhD program “Molecular
Enzymlogy” (FWF). Applied research in lipid enzymology has been performed in the Kplus
center Applied Biocatalysis at TU Graz (funding by FFG and industry).
A. Hermetter (left) and his research group
5
K. Athenstaedt, an independent group
leader at the Institute of Biochemistry
At the end of the year 2008 a grant application of Karin Athenstaedt was approved by
the Austrian Science Fund (FWF), which gave her the opportunity to continue her work
at our department as an independent researcher. With this project she set the cornerstone
to establish her own research group. Karin Athenstaedt’s work is devoted to lipid
metabolism in yeast.
In December 2009, Christian Weber, a former Assistant Professor at our department, died at
the age of 80. Christian Weber was a food chemist and has set the stage for ongoing research
in this field at our department during his active career. Moreover, he had been President of the
Alumni TU Graz 1887 for many years. We will remember him as a true representative of our
university and a kind colleague. Our deep sympathies are with his widow.

Biochemistry group
Group leader: Peter Macheroux
Secretary: Annemarie Portschy
Postdoctoral Fellow: Ines Waldner-Scott
PhD students: Alexandra Binter, Venugopal Gudipati, Tanja Knaus, Silvia Wallner
Technicians: Eva Maria Pointner (maternity leave), Steve Stipsits, Rosemarie Trenker-El-Toukhy
Alumni 2009: Sonja Sollner (PhD), Andreas Winkler (PhD), Martin Puhl (technician)
General description
The fundamental questions in the study of enzymes, the bio-catalysts of all living organisms,
revolve around their ability to select a substrate (substrate specificity) and subject this substrate to
a predetermined chemical reaction (reaction and regio-specificity). In general, only a few amino
acid residues in the "active site" of an enzyme are involved in this process and hence provide the
key to the processes taking place during enzyme catalysis. Therefore, the focus of our research is
to achieve a deeper understanding of the functional role of amino acids in the active site of
enzymes with regard to substrate-recognition and stereo- and regiospecificity of the chemical
transformation. In addition, we are also interested in substrate-triggered conformational changes
and how enzymes utilize cofactors (flavin, nicotinamide) to achieve catalysis. Towards these aims
we employ a multidisciplinary approach encompassing kinetic, thermodynamic, spectroscopic and
structural techniques. In addition, we use site-directed mutagenesis to generate mutant enzymes to
probe their functional role in the mentioned processes. Furthermore, we collaborate with our
partners in academia and industry to develop inhibitors for enzymes, which can yield important
new insights into enzyme mechanisms and can be useful as potential lead compounds in the design
of new drugs.
The methods established in our laboratory comprise kinetic (stopped-flow and rapid quench
analysis of enzymatic reactions), thermodynamic (isothermal titration microcalorimetry) and
spectroscopic (fluorescence, circular dichroism and UV/VIS absorbance) methods. We also
frequently use MALDI-TOF and ESI mass spectrometry, protein purification techniques
(chromatography and electrophoresis) and modern molecular biology methods to clone and
express genes of interest. A brief description of our current research projects is given below.
Berberine bridge enzyme & other flavin-dependent plant oxidases
Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a
pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD moiety,
which is essential for catalysis. The reaction involves the oxidation of the N-methyl group of the
substrate (S)-reticuline by the enzyme-bound flavin and concomitant formation of a carbon-carbon
bond (the “bridge”). The ultimate acceptor of the substrate-derived electrons is dioxygen, which
reoxidizes the flavin to its resting state:
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.
Recently, we have developed a new expression system for BBE (using cDNA from Eschscholzia
california, gold poppy) in Pichia pastoris, which produces large amounts of the protein (ca. 500
mg from a 10-L culture). 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). 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. These genes are
7

currently expressed in our laboratory in order to characterize the role of the enzymes in
alkaloid biosynthesis (thesis project of Silvia Wallner).
Dipeptidylpeptidase III
Dipeptidyl-peptidases III (DPPIII; EC 3.4.14.4) are Zn-dependent enzymes with molecular
masses of ca. 80-85 kDa that specifically cleave the first two amino acids from the 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. Sigrid Deller and Nina Jajcanin-Jozic have successfully expressed, purified and
characterized the recombinant yeast enzyme, and Pravas Baral in Karl Gruber’s laboratory at
the Karl-Franzens-University has elucidated the crystal structure of the protein. This work
revealed that yeast DPPIII is the first representative of a novel protein topology:
We are now focusing on structures with potential substrates or inhibitors bound in the active site.
Towards that goal, we are generating inactive mutant proteins to enable cocrystallisation with
peptide substrates. In addition, we have produced recombinant human DPPIII and are in the
process to determine the crystal structure to provide the basis for the development of potentially
useful inhibitors of the enzyme (Gustavo Arruda’s thesis project in Prof. Gruber’s laboratory
supported by Alexandra Binter).
8

Nikkomycin biosynthesis
Nikkomycins are produced by several species of Streptomyces and exhibit fungicidal, insecticidal
and acaricidal properties due to their strong inhibition of chitin synthase. Nikkomycins are
promising compounds in the cure of the immunosuppressed, such as AIDS patients, organ
transplant recipients and cancer patients undergoing chemotherapy. Nikkomycin Z (R 1 = uracil &
R 2 = OH, see below) is currently in clinical trial for its antifungal activity. Structurally,
nikkomycins can be classified as peptidyl nucleosides containing two unusual amino acids, i.e.
hydroxypyridylhomothreonine (HPHT) and aminohexuronic acid with an N-glycosidically linked
base:
Although the chemical structures of nikkomycins have been known since the 1970s, only a few
biosynthetic steps have been investigated in detail. The steps leading to the synthesis of
aminohexuronic acid are unclear. Originally, it was hypothesized that the aminohexuronic acid
moiety is generated by addition of an enolpyruvyl moiety from phosphoenolpyruvate (PEP) to
either the uridine or the 4-formyl-4-imidazolin-2-one analog at the 5’-position of ribose. This step
is then followed by rather speculative modifications to yield the aminohexuronic acid precursor. In
contrast to this hypothesis, we could recently demonstrate that UMP rather than uridine serves as
the acceptor for the enolpyruvyl moiety, a reaction catalyzed by an enzyme encoded by a gene of
the nikkomycin operon termed nikO. Furthermore, we could demonstrate that it is attached to the
3’- rather than the 5’-position of UMP. These results are very intriguing since none of the
nikkomycins synthesized possess an enolpyruvyl group in this position of the sugar moiety.
Hence, it must be concluded that the resulting 3’-enolpyruvyl-UMP is subject to rearrangement
reactions where the enolpyruvyl is detached from its 3’-position and transferred to the 5’-position
of the ensuing aminohexuronic acid moiety. Currently, nothing is known about the enzymes
involved in these putative chemical steps. However, the genes that are co-transcribed with nikO
have been reported and are designated 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 will be cloned, expressed and the proteins purified for biochemical characterization
(thesis project of Alexandra Binter).
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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 three-dimensional
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
α
β
β
α
A similar system is known from mammalian cells, where a homologous quinone reductase
(NQO1) binds to the 20S proteasome and recruits important tumor suppressor proteins such as p53
and p73α. Hence, the discovery of a homologous protein interaction in yeast provides an
interesting model system to investigate the molecular basis for protein complex formation and
regulation of proteasomal degradation of transcription factors (postdoctoral project of Ines
Waldner-Scott; thesis project of Sonja Sollner and Venugopal Gudipati).
Zn-dependent Alkylsulfatases
Yap4p
Oxidation by
e.g.
quinones
Hydrolysis of alkylsulfates is an important pathway for soil and other bacteria to mobilize sulfur.
Three classes of sulfatases have been discovered, and the recently elucidated structure of SdsA1
from Pseudomonas aeruginosa revealed that the enzyme belongs to the widely occurring family of
Zn-dependent ß-lactamases. A detailed characterization of the enzyme’s stereochemistry by the
group of Prof. Faber at the University of Graz demonstrated that this enzyme cleaves primary
alklysulfates (preferred substrate is dodecylsulfate) by retention. In other words, the hydroxyl
group attacks the sulfur atom in the course of the reaction (see reaction scheme below). Only
recently a novel enzyme activity could be identified in another Pseudomonas strain, which leads to
hydrolytic cleavage of alkylsulfates via stereoinversion, i. e. the hydroxyl group attacks the carbon
rather than the sulfur atom of the substrate (see scheme below).
10
oxidized
Lot6p
Very slow with oxygen O2 nucleus
oxygen
β
α
β
α
Yap4p
Yap4p

HO
R 1
H
R 2
HSO 4 -
Inversion
H
R 1
OH -
O
11
R 2
SO 3 -
OH -
HSO 4 -
Retention
Despite the differences in substrate preference and stereochemistry, the overall structure of this
novel enzyme (termed Pisa1 = Pseudomonas inverting sulfatase) is virtually identical to the
previously determined structure of SdsA1. 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
These amino acids are now subject of an extensive mutagenesis program to define their role in
governing substrate preference and the stereochemical course of the reaction. This project is a
close collaboration with Profs. Faber (biocatalysis) and Wagner (structure determination) from the
University of Graz (thesis project of Tanja Knaus in our laboratory and Markus Schober in Prof.
Faber’s laboratory).
Doctoral theses completed
Tyr/Ser
Met/Ser
Met/Ser
Tyr/Ser
Leu/Pro
Sonja Sollner: Function and mechanism of Lot6p from Saccharomyces cerevisiae, a flavindependent
quinone reductase
Flavin-dependent NAD(P)H:quinone oxidoreductases have been reported from several
mammalian sources. These enzymes have been thought to have a protective effect against
quinone-related oxidative cell damage through their unique ability to transfer two electrons to a
H
R 1
Ala/Ile
OH
R2

quinone thereby catalyzing the formation of a fully reduced hydroquinone. Interestingly, those
enzymes are found in solid tumours at increased levels and thus can be used to target the tumour
cell through bioreductive activation of prodrugs. Here the identification and characterization of
Lot6p, a soluble, flavin-dependent quinone reductase from Saccharomyces cerevisiae is described.
It is shown that Lot6p is capable of reducing a variety of substrates at the expense of either NADH
or NADPH via a ping-pong bi bi reaction mechanism. Recent studies using human quinone
reductases demonstrated that this enzyme binds to the 20S proteasome and affects the lifetime of
transcription factors, such as tumour suppressor p53, by inhibiting their intracellular degradation.
It could be shown that Lot6p is physically associated with the yeast proteasome. Furthermore,
quinone reductase lacking the flavin cofactor was unable to bind to the proteasome indicating that
only the holo-protein can form a protein complex. Interaction of Lot6p with the proteasome is
independent of the redox state of the flavin. However, reduction of the FMN cofactor triggers
association of the complex with Yap4p, a transcription factor involved in the oxidative stress
response. Moreover, Yap4p was detected in the nucleus of quinone stressed yeast cells, but not in
the nucleus of non-stressed wild-type cells. This finding suggests that stress causes the release of
the transcription factor from the protein complex resulting in concomittant relocalisation to the
nucleus. Sequestration of a transcription factor by a quinone-reductase-proteasome complex
discovered here is similar to findings in mammalian cells.
Our data also reveal that the redox state of the flavin cofactor controls recruitment of the
transcription factor Yap4p to the quinone reductase-proteasome complex. This would imply that
stability and localization of Yap4p are under direct redox control of the Lot6p-proteasome
complex. Under reducing conditions (when an excess of NAD(P)H is present in the cell), Yap4p is
bound to the quinone reductase-proteasome system, virtually in a stand-by position. In the
presence of quinones (i.e., when oxidative stress is applied to the cell), Yap4p is released from the
complex and rapidly relocalizes to the nucleus where it is now able to switch on stress-related
target genes.
Andreas Winkler: Structure-function studies on berberine bridge enzyme from the California
poppy (Eschscholzia californica)
Berberine bridge enzyme catalyzes the oxidative conversion of (S)-reticuline to (S)-scoulerine by
formation of a C-C bond between the N-methyl group and the phenolic moiety of the substrate.
This reaction leads to ring closure and is unique to the biosynthesis of benzophenanthridine
alkaloids with no direct equivalent in organic chemistry. We therefore set out to characterize the
molecular mechanism by means of a functional and structural analysis of the enzyme. The
biochemical characterization of the flavin cofactor revealed that it is covalently linked to the
protein backbone via two amino acids (His-104 and Cys-166). At the time of discovery this
unusual bicovalent mode of FAD attachment was just recently identified during the structural
analysis of a distantly related enzyme. We could show that this dual mode of cofactor linkage
increases the redox potential to the highest value reported for flavoenzymes so far (+132 mV). The
analysis of mutant proteins lacking each of these linkages (H104A and C166A) confirmed their
contribution to the elevated redox potential (-100 and -80 mV difference, respectively) but in
addition also demonstrated their importance for correct positioning of the substrate in the active
site allowing efficient turnover. These conclusions were supported by the elucidation of the crystal
structures of wild type BBE as well as those of the mutant proteins H104A and C166A.
Comparing the active site architecture between these structures demonstrated that the overall
positioning of the cofactor is only slightly affected by the removal of the covalent linkages, while
surrounding amino acids respond significantly to replacement of either His-104 or Cys-166.
Especially the flexibility of Trp-165, which plays an important role during the conversion of
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substrate to product, was reduced extensively. Its substantial conformational change during
turnover was observed in soaking experiments with (S)-reticuline leading to complex structures
with both substrate and product bound. These structures also allowed the identification of potential
active site amino acids involved in the catalytic mechanism. Determination of kinetic rates for
three active site protein variants identified a glutamate residue (Glu-417) to be essential for
cofactor reduction and formation of the berberine bridge. Based on our findings, we proposed a
catalytic mechanism for BBE that entails proton abstraction of the aromatic hydroxyl group
leading to concerted C-C coupling accompanied by hydride transfer from the N-methyl group to
the N5 atom of the FAD cofactor.
International cooperations
M. Abramic, Ruder Boskovic Institute Zagreb, Croatia
F. Hollmann, Delft University of Technology, The Netherlands
M. Groll, Technical University Munich, Germany
T. Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A.
S.-H. Liaw, National Yang-Ming University, Taipei, Taiwan
B. A. Palfey, University of Michigan, Ann Arbor, U.S.A.
I. Tews, Universität Heidelberg, Germany
Research projects
FWF P19858: “Enzymes of nikkomycin biosynthesis”
FWF-Doktoratskolleg “Molecular Enzymology”
WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49 family”
Publications
1) Sollner, S., Schober, M., Wagner, A., Prem, A., Lorkova, L., Palfey, B. A., Groll, M.,
and Macheroux, P.: Quinone reductase acts as a redox switch of the 20S yeast
proteasome, EMBO Reports, 2009, 10:65-70.
2) Mueller, N. J., Stueckler, C., Hall, M., Macheroux, P., Faber, K.: Epoxidation of
conjugated C=C-bonds and sulfur-oxidation of thioethers mediated by NADH:FMNdependent
oxidoreductases, Org. Biomol. Chem., 2009, 7:1115-1119.
3) Wallner, S., Neuwirth, M., Flicker, K., Tews, I., and Macheroux, P.: Dissection of
contributions from invariant amino acids to complex formation and catalysis in the
heteromeric pyridoxal-5’-phosphate synthase complex, Biochemistry, 2009, 48:1928-
1935.
4) Winkler, A., Motz, K., Riedl, Sabrina, Puhl, M., Macheroux, P., Gruber, K.: Structural
roles of bicovalent flavinylation in berberine bridge enzyme, J. Biol. Chem., 2009,
284:19993-20001.
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5) Neuwirth, M., Strohmeier, M., Windeisen, V., Wallner, S., Deller, S., Rippe, K.,
Sinning, I., Macheroux, P., and Tews, I.: X-ray crystal structure of Saccharomyces
cerevisiae Snz1 provides insight into the oligomeric nature of PLP synthases, FEBS
Lett., 2009, 583:2179-2186.
6) Sollner, S., Macheroux, P.: New roles of flavoproteins in molecular cell biology: An
unexpected role for quinone reductases as regulators of proteasomal degradation, FEBS
J., 2009, 276:4313-4324.
7) Sollner, S., Durchschlag, M., Fröhlich, K.-U., Macheroux, P.: The redox-sensing
quinone reductase Lot6p acts as an inducer of yeast apoptosis, FEMS Yeast Res., 2009,
9:885-891.
8) Winkler, A., Puhl, M., Weber, H., Kutchan, T. M., Gruber, K., Macheroux, P.:
Berberine bridge enzyme catalyzes the six-electron oxidation of (S)-reticuline to
dehydroscoulerine, Phytochemistry, 2009, 70:1092-1097.
9) Binter, A., Staunig, N., Jelesarov, I., Lohner, K., Palfey, B. A., Deller, S., Gruber, K.,
Macheroux, P.: A single intersubunit salt-bridge affects oligomerization and catalytic
activity in a bacterial quinone reductase, FEBS J., 2009, 276:5263-5274.
10) Breithaupt, C., Kurzbauer, R., Schaller, F., Schaller, A., Huber, R., Macheroux, P., and
Clausen, T.: Structural basis of substrate specificity of plant 12-oxophytodienoate
reductases, J. Mol. Biol., 2009, 392:1266-1277.
11) Sollner, S., Deller, S., Macheroux, P. and Palfey, B. A.: Mechanism of flavin reduction
and oxidation in the redox sensing quinone reductase Lot6p from Saccharomyces
cerevisiae, Biochemistry, 2009, 48:8636-8643.
12) Grau, M. M., van der Toorn, J., Otten, L. G., Macheroux, P., Taglieber, A., Zilly, F. E.,
Arends, W. C. E., Hollmann, F.: Photoenzymatic reductions of C=C double bonds, Adv.
Synth. Catal., 2009, 351:3279-3286.
Award
Styrian Research Award 2009 to Peter Macheroux for his work on “Redox-regulated degradation
of proteins by the quinone reductase-proteasome complex”.
14

Cell Biology Group
Group leader: Günther Daum
Graduate students: Tibor Czabany, Melanie Connerth, Sona Rajakumari, Karlheinz Grillitsch,
Miroslava Spanova, Susanne Horvath, Martina Gsell, Vid V. Flis
Diploma student: Sabine Zitzenbacher
Technician: Claudia Hrastnik
Visiting scientists: Sabina Tavares, Fluxome Company, Lyngby, Denmark
General description
The existence of functional organelles is the basis for regulated processes within a cell. To
sequester organelles from their environment, membranes are required which not only protect
the interior of the organelles but also govern communication with the rest of the cell.
Therefore, it is very important to understand the biogenesis and maintenance of biological
membranes. To study this question our laboratory makes use of the yeast as a well established
experimental system. Our studies are focused on the synthesis of lipids, their storage and their
assembly into organelle membranes. We make use of the advantages of this experimental
system to combine biochemical, molecular and cell biological methods addressing problems
of lipid metabolism, lipid depot formation and membrane biogenesis.
The majority of yeast lipids are synthesized in the endoplasmic reticulum (ER) with some
significant contributions of mitochondria, the Golgi and the so-called lipid particles. Other
subcellular fractions are devoid of lipid-synthesizing activities. Spatial separation of lipid
biosynthetic steps and lack of lipid synthesis in several cellular membranes necessitate
efficient transfer of lipids from their site of synthesis to their proper destination(s). The
maintenance of organelle lipid profiles requires strict coordination of biosynthetic and
translocation activities.
Specific aspects studied recently in our laboratory are (i) assembly and homeostasis of
phosphatidylethanolamine in yeast organelle membranes with emphasis on peroxisomes and
the plasma membrane, (ii) neutral lipid storage in lipid particles/droplets and mobilization of
these depots with emphasis on the involvement of lipases and hydrolases, and (iii)
characterization of organelle membranes from the industrial yeast Pichia pastoris.
Phosphatidylethanolamine, a key component of yeast organelle membranes
Work from our laboratory and from other groups had shown that phosphatidylethanolamine
(PE), one of the major phospholipids of yeast membranes, is highly important for cellular
function and cell proliferation. PE synthesis in the yeast is accomplished by a network of
reactions including (i) synthesis of phosphatidylserine (PS) in the endoplasmic reticulum,
(ii) decarboxylation of PS by mitochondrial phosphatidylserine decarboxylase 1 (Psd1p) or
(iii) Psd2p in a Golgi/vacuolar compartment, (iv) the CDP-ethanolamine pathway (Kennedy
pathway) in the endoplasmic reticulum, and (v) the lysophospholipid acylation route
catalyzed by Ale1p. To obtain more insight into biosynthesis, assembly and homeostasis of
PE single and multiple mutants bearing defects in the respective pathways can be used.
15

Previous investigations in our laboratory were aimed at the molecular biological
identification of novel components involved in PE homeostasis of the yeast Saccharomyces
cerevisiae. For this purpose, a number of genetic screenings were performed. To obtain a
global view of the role of PE in the cell and to study the effects of an unbalanced PE level we
recently subjected a psd1∆ deletion mutant and the corresponding wild type to DNA
microarray analysis and examined genome-wide changes in gene expression caused by a
PSD1 deletion. Comparison of the gene expression pattern of the psd1∆ mutant with the
wild-type led to the identification of 55 differentially expressed genes. Grouping of these
genes into functional categories revealed that PE formation by Psd1p influenced the
expression of genes involved in diverse cellular pathways including transport, carbohydrate
metabolism and stress response. Most of the proteins identified are present in the cytosol
followed by the nucleus, cellular membranes, the cell wall and mitochondria. Moreover, this
genome-wide analysis identified a number of gene products with unknown function which
are currently subjected to detailed molecular analysis addressing PE homeostasis in the yeast.
16
Figure 1:
Three-dimensional reconstruction of
yeast cells grown on oleic acid.
Transmission electron micrographs of
ultrathin sections (A, C; bar = 1µm)
and corresponding 3D reconstructions
of serial sections (B, D) of a
chemically fixed wild type cell are
shown. Cells shown in A and B were
grown on YPD, and cells shown in C
and D were grown on oleic acid to
induce formation of peroxisomes. CW
cell wall; ER endoplasmic reticulum;
LP lipid particle; M mitochondrion; N
nucleus; V vacuole; PX peroxisomes.
By courtesy of G. Zellnig, KFUG.
To understand cellular PE homeostasis in some more detail, we performed experiments
defining traffic routes of PE within the yeast cell. In recent studies, we investigated
peroxisomes and the plasma membrane as destinations for PE distribution. These two
membranes have in common their lack of capacity to synthesize PE. We employed yeast
mutants bearing defects in the different pathways of PE synthesis and demonstrated that PE
formed though all four pathways can be supplied to peroxisomes and to the plasma
membrane. The fatty acid composition of peroxisomal and plasma membrane phospholipids
was mostly influenced by the available pool of total species synthesized, although a certain
balancing effect was observed regarding the assembly of PE species into the two target
compartments. We assume that the phospholipid composition of peroxisomes and the plasma
membrane is mainly affected by the synthesis of the respective components and subject to
equilibrium, and to a lesser extent affected by specific transport and assembly processes.

Organelle association and membrane contact between organelles (Fig. 1) may be a relevant
mechanism for lipid transport between organelles, but components governing this process
and related events have to be studied in future investigations.
Growth of yeast on oleic acid not only induces formation of peroxisomes, but also leads to an
intense synthesis of triacylglycerols which are stored in lipid particles (see below). The link
between neutral lipid metabolism and peroxisome proliferation is subject to current
investigations with emphasis on the role of enzymes involved. These studied are related to
the possible lipotoxic effect of fatty acids in yeast. Most recently we also studied the link
between PE metabolism and neutral lipid storage. For this purpose we analyzed lipids from
strains bearing defects in PE synthesis. These analyses showed that a mutant bearing a defect
in the CDP-ethanolamine pathway had a decreased level of triacylglycerols (TAG). The
molecular reason for this finding is currently under investigation.
Neutral lipid storage in lipid particles and mobilization
Yeast cells have the capacity to store neutral lipids TAG and STE (steryl esters) in subcellular
structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized
and serve as building blocks for membrane biosynthesis. In an ongoing project of our
laboratory, we focused on the characterization of enzymatic steps which lead to the
mobilization of TAG and STE depots.
In Saccharomyces cerevisiae, the three STE hydrolases Tgl1p, Yeh1p and Yeh2p contribute
differently to STE mobilization from their site of storage, the lipid particles. In a biochemical
study we investigated enzymatic and cellular properties of these three hydrolytic enzymes.
Using the respective single, double and triple deletion mutants and strains overexpressing the
three enzymes, we demonstrated that each STE hydrolase exhibits certain substrate
specificity. Interestingly, disturbance in STE mobilization also affects sterol biosynthesis in a
type of feedback regulation. We also showed that sterol intermediates stored in STE and set
free by STE hydrolases are recycled to the sterol biosynthetic pathway and converted to the
final product, ergosterol. This recycling implies that the vast majority of sterol precursors are
transported from lipid particles to the ER where sterol biosynthesis is completed. Ergosterol
formed through this route is then supplied to its subcellular destinations, especially the plasma
membrane. Only a minor amount of sterol precursors set free from STE are randomly
distributed within the cell after cleavage. In conclusion, STE storage and mobilization
although dispensable for yeast viability contribute markedly to sterol homeostasis and
distribution.
A major focus of our neutral lipid project was the biochemical characterization of the three
yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had
demonstrated that deletion of TGL3 encoding the major yeast TAG lipase resulted in
decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids,
especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG
fraction. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we
carried out biochemical experiments with wild type and deletion strains bearing defects in
Tgl3p, Tgl4p and Tgl5p. We demonstrated that tgl mutants had a lower level of sphingolipids
and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG
accumulation in these mutant cells resulted in reduced amounts of phospholipids and
sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected
17

the metabolic flux of long chain fatty acids and very long chain fatty acids required for
sphingolipid and glycerophospholipid synthesis. Moreover, activity and expression levels of
fatty acid elongases, Elo1p and Elo2p, were enhanced as a consequence of reduced TAG
lipolysis emphasizing the link to sphingolipid formation. Finally, the pattern of
phosphatidylcholine (PC), PE and PS molecular species was altered in tgl deletion strain
underlining the important role of TAG turnover in maintenance of the pool size and
remodelling of complex membrane lipids. This study shed new light on the physiological role
of TAG lipases in yeast and in general.
Figure 2: Domains of Tgl3p (from Rajakumari et al., 2010, in press)
Even more surprising evidence was obtained when the enzymology of the three TAG lipases
Tgl3p, Tgl4p and Tgl5p was studied in some detail. Motif search analysis indicated that Tgl3p
and Tgl5p did not only contain the TAG lipase but also an acyltransferase motif (Figure 2).
Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and
overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained
with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed,
both enzymes did not only exhibit lipase activity but also catalyzed acylation of
lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using
variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two
enzymatic activities act independently of each other. We also showed that Tgl3p is important
for efficient sporulation of yeast cells, but rather through its acyltransferase than lipase
activity. These results demonstrated that yeast Tgl3p and Tgl5p play a dual role in lipid
metabolism contributing to both anabolic and catabolic processes.
In another study, we demonstrated that the yeast TAG lipase Tgl4p, the functional ortholog of
adipose TAG lipase (ATGL), catalyzes multiple functions in lipid metabolism. An extended
domain and motif search analysis revealed that Tgl4p bears not only a lipase consensus
domain but also a conserved motif for calcium independent phospholipases A2 (PLA2). We
showed that Tgl4p exhibits TAG lipase, STE hydrolase and PLA2 activities, but also
catalyzes acyl-CoA dependent acylation of lysophosphatidic acid (LPA) to phosphatidic acid.
Heterologous overexpression of Tgl4p in Pichia pastoris increased total phospholipid and
specifically phosphatidic acid synthesis. Moreover, deletion of TGL4 in Saccharomyces
cerevisiae showed an altered pattern of PC and phosphatidic acid molecular species.
Altogether, our data suggested that yeast Tgl4p functions as hydrolytic enzyme in lipid
degradation, but also contributes to fatty acid channelling and phospholipid remodelling.
Several years ago we identified through a mass spectrometric approach the major lipid
particle proteins of Saccharomyces cerevisiae. This approach was a milestone in the field
because it identified a number of new gene products and their function and provided valuable
hints for processes associated with lipid particles. We realized, however, that this study was
18

incomplete and did not provide the complete picture of the yeast lipid particle proteome. For
this reason, a more precise lipid particle proteome analysis was initiated in collaboration with
M. Karas from the Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe
University, Frankfurt, Germany. This proteome study was combined with a lipidomics
investigation that was performed in collaboration with H. Köfeler from the Center for Medical
Research, Medical University of Graz, Austria. In this study, we compared lipid particle
components from cells which were grown on glucose or on oleic acid. This approach
identified a number of lipid particle proteins that were already known but also some novel
polypeptide candidates. Detailed investigations of these proteins are currently in progress. We
also realized through this approach that there were some differences in the lipid particle
proteome from cells grown on glucose or oleic acid. Finally, mass spectrometric analyses
revealed marked differences in the lipidome of lipid particles from cells grown on the two
different carbon sources. This study sets the stage for further investigation of protein-lipid
interaction on the surface of lipid particles and provides basic evidence for the coordinated
biosynthesis of lipid and protein components from lipid particles.
Another important aspect of this study was the physiological relevance of TAG and STE
depot formation. Therefore, attempts were made to understand cell biological consequences of
dysfunctions in neutral lipid storage. Previously, it has been reported that yeast cells lacking
neutral lipid depots appear to become apoptotic. More recently, we and others were able to
demonstrate that lipid depot formation plays an important role especially when yeast cells are
stressed in the presence of exogenous fatty acids. However, it appears that an adaptation to
this stress situation occurs which helps to overcome the deleterious effect of this lipotoxic
stress. Changes in the lipid pattern caused by cultivation of yeast cells on oleic acid are
currently investigated in some detail.
In a previous study we had observed that variation of the yeast lipid particle composition by
deletion of either TAG or STE synthesizing enzymes led to changes in the protein pattern on
the lipid particle surface. In particular we realized that Erg1p, which is present at large
amounts on lipid particles of wild type cells, loses stability in strains missing STE. During an
ongoing project we performed stability and topology studies with different variants and
constructs of Erg1p to elucidate embedding of this protein in the lipid particle surface
membrane. This problem is related to the general question how lipid particle proteins are
anchored in the monolayer membrane of the particle and/or interact with the hydrophobic core
of the compartment. Although the evidence obtained so far is only preliminary we believe that
the lipid status of lipid particles has a direct influence on the protein equipment of this
compartment. This link may be related to the biogenesis of lipid particles.
Another potential non-polar storage lipid is squalene. This component belongs to the group of
isoprenoids and is precursor for the synthesis of sterols, steroids and ubiquinons. In the yeast,
the amount of squalene can be increased by varying culture conditions or by genetic
manipulation. As an example, in strains deleted of HEM1 squalene accumulates and is stored
mainly in lipid particles. Interestingly, a heme deficient dga1∆lro1∆are1∆are2∆ quadruple
mutant (Qhem1∆) which is devoid of the classical storage lipids, TAG and SE, accumulates
substantial amounts of squalene in cellular membranes, especially in microsomes and the
plasma membrane. The fact that Qhem1∆ does not form lipid particles suggests that
biogenesis of these storage particles cannot be initiated by squalene which is synthesized in
the ER similar to TAG and SE. This result also indicates that squalene accumulation, at least
under the conditions tested, is not lipotoxic. Finally, our experiments demonstrate that
squalene incorporated into organelle membranes does not compromise cellular function.
19

Pichia pastoris organelles
The yeast Pichia pastoris is an important experimental system for heterologous expression of
proteins. Nevertheless, surprisingly little is known about organelles of this microorganism.
For this reason, we started a systematic biochemical and cell biological study to establish
standardized methods of Pichia pastoris organelle isolation and characterization. Recent work
focused on the biochemical characterization of mitochondrial membranes and the plasma
membrane from Pichia pastoris grown on different carbon sources. The major aim of this
project was the qualitative and quantitative analysis of phospholipids, fatty acids and sterols
from both types of membranes, when Pichia pastoris was grown on different carbon sources.
We also started to study selected lipid synthesizing enzymes from Pichia pastoris with
emphasis on the PE biosynthetic machinery. We identified two Pichia pastoris PS
decarboxylases (PSD) encoded by genes homologous to PSD1 and PSD2 from
Saccharomyces cerevisiae (Figure 3). Using Pichia pastoris psd1 and psd2 mutants we
investigated the role of these two gene products in PE synthesis and the effect of their
deletions on cell growth. Mitochondrial Psd1p is the major enzyme for PE synthesis in Pichia
pastoris. Deletion of PSD1 caused a loss of PSD activity in mitochondria and a severe growth
defect on minimal media which was partially cured by supplementation with ethanolamine.
This result indicated that the CDP-ethanolamine but not the Psd2p pathway provided
sufficient PE for cell viability in a psd1 background. Under these conditions, however,
cellular and mitochondrial PE levels were dramatically reduced. Fatty acid analysis from wild
type, psd1 and psd2 demonstrated the selectivity of both PSDs in vivo for the synthesis of
unsaturated PE species. Short chain saturated (16:0) PE species were preferentially imported
into mitochondria irrespective of psd1 or psd2 deletions. Thus, biosynthesis and subcellular
distribution contribute to PE homeostasis in mitochondria of Pichia pastoris.
.
Figure 3:
Phylogenetic tree of PSD sequences from different organisms. The tree was constructed using
the neighbor joining (NJ) method of the CLUSTALW program.
20

Diploma Thesis completed
Sabine Zitzenbacher: Characterization of organelles from the yeast Pichia pastoris
Characterizations of cell organelles from organisms start with a successful isolation of the
organelle of interest. Isolation protocols are available, but they usually have to be optimized.
A problem of each organelle preparation is cross-contamination with other cellular organelles
in the isolated organelle fraction. Here we tested the quality of preparation protocols for
mitochondria and microsomes, plasma membrane and vacuoles, used for the strain Pichia
pastoris. Isolated fractions were analysed by Western blot analysis and functional enzyme
assays. Results of isolated mitochondrial and microsomal fractions showed a clear enrichment
of mitochondria as well as microsomes. In the mitochondrial fraction a minor ER
contamination was observed, whereas the microsomes fractions were free from crosscontamination.
Analysis of the isolated plasma membrane fraction showed enrichment of
plasma membrane without any cross contamination with other organelles. Both preparation
protocols have proven to be well established for Pichia pastoris. The last protocol
investigated showed an enrichment of vacuoles in the isolated fraction, but the vacuolar
fraction also showed a high contamination with mitochondria. Thus, the protocol has to be
modified to get an isolated vacuolar fraction without any or with only little crosscontamination.
In the past, different cell organelles, such as peroxisomes, mitochondria,
microsomes and plasma membrane from Pichia pastoris were characterized. The missing
organelle is the Golgi apparatus. Here we established an isolation protocol for Golgi isolation
from Pichia pastoris. The obtained Golgi fraction was analysed by Western Blot and
enzymatic assays. Applying this new preparation protocol we showed an enrichment of Golgi
in the isolated fraction largely free of cross-contamination with ER.
Doctoral Thesis completed
Tibor Czabany: Cell biology of neutral lipid synthesis in yeast Saccharomyces cerevisiae
Previously, four acyltransferases involved in the synthesis of neutral lipids (NL),
triacylglycerols (TAG) and steryl esters (SE), have been identified. Dga1p and Lro1p esterify
DAG to produce TAG, and Are1p and Are2p are responsible for SE synthesis. After the NL
have been synthesized they are assembled in specialized organelles called lipid particles (LP).
The hydrophobic core of LP containing TAG and SE is separated from the hydrophilic
cytosol by a phospholipid monolayer. The present study is focused on elucidation of the
detailed structure using a set of triple mutants (TM) which contain only one of the four
acyltransferases active. Using these mutants we were able to investigate the influence of
individual enzymes on LP composition and structure. It was demonstrated that the four
acyltransferases act independently of each other and are able to synthesize individually a
sufficient amount of neutral lipids for LP biogenesis. Furthermore, TM and the quadruple
mutant, lacking all four acyltransferases, were also used for studies of NL synthesis when
fatty acids were supplied as the only carbon source. Under these conditions, fatty acids enter
the cell and accumulate at high amounts which leads to disturbed lipid homeostasis. Changes
in lipid metabolism caused by the lack of individual acyltransferase are described, especially
the influence of excess fatty acids on neutral lipid synthesis. It was shown that yeast cells
grown on oleic acid upregulated TAG synthesis and down regulated esterification of sterols.
Detailed analysis of SE synthase revealed that the down regulation of this activity was not by
transcriptional control but rather by direct inhibition of the enzyme in its membrane
21

environment. In summary, this work describes biochemical and cell biological functions of
neutral lipid synthesizing acyltransferases and the link to neutral lipid storage in LP.
International Cooperations
J. Cregg, Keck Graduate Institute of Applied Life Sciences, Claremont, CA, USA
I. Hapala, Slovak Academy of Sciences, Institute of Animal Biochemistry and Genetics,
Ivanka pri Dunaji, Slovak Republic
I. Feussner, Plant Biochemistry, Albrecht-von-Haller-Institute of Plant Sciences, Georg-
August University Göttingen, Germany
R. Erdmann, Institute of Physiological Chemistry, Ruhr-University Bochum, Germany
T. Höfken, Institute of Biochemistry, Christian Albrecht University, Kiel, Germany
M. Karas, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University,
Frankfurt, Germany
R. Rajasekharan, Department of Biochemistry, Indian Institute of Science, Bangalore, India
A. S. Sandelius, Department of Plant and Environmental Sciences, University of Gothenburg,
Sweden
T. Höfken, Institute of Biochemistry, Christian Albrecht University, Kiel, Germany
Research Projects
FWF 17321: Phosphatidylethanolamine of yeast mitochondria
FWF 21429: Phosphatidylserine decarboxylase
FWF 18857: Neutral lipid storage and mobilization in the yeast Saccharomyces cerevisiae
FWF L-178 (Translational Research): Characterization of organelles from the yeast Pichia
pastoris
FWF PhD Program: Molecular Enzymology
Invited Lectures
1. G. Daum
University of Bochum, Germany
Turnover of neutral lipids and channeling of lipid storage components in the yeast
April 2009
2. G. Daum, T. Czabany, A. Wagner, K.H. Grillitsch, M. Connerth and S. Rajakumari
Turnover of neutral lipids and channeling of lipid storage components in the yeast
9th Yeast Lipid Conference, Berlin, Germany, 21-23 May 2009
3. G. Daum, T. Czabany, A. Wagner, K.H. Grillitsch, M. Connerth and S. Rajakumari
Turnover of neutral lipids and channeling of lipid storage components in the yeast
27 th ISSY: Pasteur’s legacy – Yeast for Health and Biotechnologies, Paris, France, 26-29
August 2009
22

Publications
1. Wagner, A., Grillitsch, K., Leitner, E. and Daum, G.
Mobilization of steryl esters from lipid particles of the yeast Saccharomyces cerevisiae.
Biochim. Biophys. Acta 1791 (2009) 118-124
2. Schuiki, I., and Daum, G.
Phosphatidylserine decarboxylases, key enzymes of lipid metabolism
IUBMB Life 61 (2009) 151-162
3. Wriessnegger, T., Leitner, E., Belegratis, M. R., Ingolic, E. and Daum, G.
Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris.
Biochim. Biophys. Acta 1791 (2009) 166-172
4. Rosenberger, S., Connerth, M., Zellnig, G. and Daum, G.
Phosphatidylethanolamine synthesized by three different pathways is supplied to
peroxisomes of the yeast Saccharomyces cerevisiae.
Biochim. Biophys. Acta 1791 (2009) 379-387
5. Wriessnegger, T, Sunga , A. J., Cregg J. M. and Daum, G.
Identification of phosphatidylserine decarboxylases 1 and 2 from Pichia pastoris
FEMS Yeast Res. 9 (2009) 911-922
6. Ghosh, A.K., Chauhan, N., Rajakumari, S., Daum, G. and Rajasekharan, R.
At4g24160, A Soluble Acyl-Coenzyme Dependent Lysophosphatidic Acid
Acyltransferase
Plant Physiol. 151 (2009) 869-881
7. Connerth, M., Grillitsch, K., Köfeler, H. and Daum, G.
Analysis of Lipid Particles from Yeast
Methods Mol. Biol. 579 (2009) 359-374
8. Lin, M., Grillitsch, K., Daum, G., Just, U. and Höfken, T.
Modulation of sterol homeostasis by the Cdc42p effectors Cla4p and Ste20p in the yeast
Saccharomyces cerevisiae.
FEBS J. 276 (2009) 7253-7264
9. Rajakumari, S. and Daum, G.
Janus-faced enzymes yeast Tgl3p and Tgl5p catalyze lipase and acyltransferase reactions
Mol. Biol. Cell (2009) in press
10. Schuiki, I., Schnabl, M., Czabany, T., Hrastnik, C. and Daum, G.
Phosphatidylethanolamine synthesized by four different pathways is supplied to the
plasma membrane of the yeast Saccharomyces cerevisiae.
Biochim. Biophys. Acta (2009) in press
11. Spanova, M., Czabany, T., Zellnig, G., Leitner, E., Hapala, I. and Daum, G.
Effect of lipid particle biogenesis on the subcellular distribution of squalene in the yeast
Saccharomyces cerevisiae.
J. Bio. Chem. (2009) in press
Award
Sona Rajakumari
BBA Young Speaker Award, 50th International Conference on Bioscience of Lipids (ICBL),
Regensburg, Germany, September 2009
23

Molecular Biology Group
Karin Athenstaedt
Triacylglycerol synthesis in the oleaginous yeast Yarrowia lipolytica
The oleaginous yeast Yarrowia lipolytica has an outstanding capacity to accumulate huge
amounts of triacylglycerols (TAG). Along with other neutral lipids, TAG are stored in socalled
lipid particles. The structure of these cell compartments is rather simple. Mainly TAG
and steryl esters are forming a hydrophobic core which is surrounded by a phospholipid
monolayer with few proteins embedded. By growing Yarrowia lipolytica cells in media
containing, e.g., industrial fats or glycerol as a carbon source, the amount of TAG can be
increased up to 40% of cell dry weight. This ability leads to its application in biotechnological
processes such as single cell oil production or production of nutrients enriched in essential
fatty acids which can serve as nutritional complements. However, Yarrowia lipolytica may
also serve as a model organism to study lipid turnover in adipocytes, since not only the ability
to store excessive amounts of TAG in lipid particles but also the composition of this cell
compartment resembles adipocytes of higher eukaryotes. Despite these potentials of Yarrowia
lipolytica information about TAG (lipid) metabolism in this yeast is rather limited. Thus, we
started to investigate the proteome of Yarrowia lipolytica for proteins involved in TAG
synthesis. Homology searches with TAG synthases of other eukaryotes as queries highlighted
two candidate gene-products of the oleaginous yeast potentially catalyzing the formation of
TAG. A decreased amount of TAG in mutant cells defective in these candidate genes already
pinpointed to a function of these polypeptides in TAG formation. To investigate whether
these candidate genes encode true TAG synthases these genes were heterologously expressed
in cells of a Saccharomyces cerevisiae mutant defective in neutral lipid synthesis and as a
consequence lacking lipid particles (Fig. 1).
A B
C D
Figure 1: Restoration of lipid particle formation upon heterologous expression of Yarrowia
lipolytica TAG synthase candidates in mutant cells of the budding yeast Saccharomyces
cerevisiae lacking lipid particles.
In contrast to the negative control (B; mutant + empty plasmid), lipid particles are formed in
mutant cells transformed with plasmids bearing either of the respective candidate genes of
Yarrowia lipolytica (C, D). Panel A shows lipid particle formation in a wild-type cell of
Saccharomyces cerevisiae. Lipid particles are indicated by arrows. Size bar: 10 µm.
Fluorescent microscopic inspection and lipid analyses of the respective mutants clearly
demonstrated that these Yarrowia lipolytica genes encode true TAG synthases. The
24

characteristics of these two TAG synthases of the oleaginous yeast are currently under
investigation.
Is the function of Nte1p conserved from yeast to man?
Neuropathy target esterase (NTE) was originally identified as the target of
organophosphorous esters (OP) present in many pesticides as well as warfare agents.
Toxifications with OPs are causing delayed neuropathy in man and some other vertebrates.
The syndrome of the organophosphorous-induced delayed neuropathy (OPIDN) is
characterized by paralysis of the lower limbs and degeneration of long axons in the spinal
cord. These effects are common responses of neurons in metabolic disorders (e.g., diabetes),
toxic states and advanced age. Neither NTE nor its homologs in other organisms are closely
related to any other esterase or protease. The importance of NTE can be seen by the fact that
mice lacking this protein die as early embryos, and inactivation of SWS (swiss cheese), the
homolog of NTE in the fruit fly Drosophila melanogaster, leads to a progressive degeneration
of the nervous system, which can be observed as big “holes” in the brain of the fly.
Wild type, 21d sws, 8d
Figure 2: “Holes” in the brain of a sws-mutant of the fruit fly Drosophila melanogaster.
Heterologous expression of the murine NTE can completely substitute SWS in Drosophila,
demonstrating the functional conservation of this protein. A homolog of NTE is also present
in the yeast Saccharomyces cerevisiae encoded by NTE1. In contrast to higher eukaryotes,
deletion of this protein in the model organism yeast does not result in a phenotype under
several standard conditions tested. However, due to the presence of this polypeptide in a
single cell organism, NTE has to be involved in a more general context as in development,
neural survival and brain integrity. All efforts to identify the biological function, substrate,
and interaction partners of NTE in higher eukaryotes have failed so far. In contrast, Nte1p of
the yeast has been reported to exhibit phospholipase B activity, thus indicating a similar
function of the respective counterparts of higher eukaryotes. The question arises whether
Nte1p of Saccharomyces cerevisiae is a true member of the NTE protein family and thus
suitable as a model for unraveling the biological function of the NTE protein family. In vitro,
the functional conservation between NTE of higher eukaryotes and Nte1p of the yeast has
already been demonstrated. But is this also true in vivo? For answering this question, I
expressed NTE1 of the yeast heterologously in the fruit fly Drosophila melanogaster. A
functional compensation would be indicated by reversion of the phenotype of an sws mutant
25

fly, i.e., observable as a decrease in the size and/or number of “holes” in the brain of
Drosophila. Several different mutant lines were constructed expressing the yeast NTE1 to
different extent and in different tissues. Subsequently, the brains of these mutants were
analyzed by fluorescent microscopy for the presence of “holes”. Any result from full
reversion of the phenotype to no effect has been considered, however, the phenotype was even
worse in the transfectants, showing a higher number of “holes”. In addition, the life span of
the sws-mutants expressing NTE1 of yeast was significantly reduced compared to control
flies. These results indicate that there is no functional conservation among the NTE protein
family in vivo. However, investigations what caused this rather striking effect provided some
evidence that Nte1p interacts with enzymes of the lipid metabolism and forms a protein
complex with at least one more polypeptide. Results of experiments performed with
Drosophila indicated that this is also true for Sws of the fruit fly. Furthermore, mutations in
certain lipid synthesizing pathways led to smaller and fewer holes in the brain of a sws-mutant
fly, indicating that the phenotype of the sws mutant is caused by major changes in the lipid
pattern. Analysis of the lipid pattern of the respective mutant flies is currently under
investigation and will reveal which lipid parameters are required for normal development of
the nervous system. This will provide valuable information for potential targets to compensate
effects caused by neurons in metabolic disorders (e.g., diabetes), toxic states and advanced
age.
International Cooperations
T. Chardot and J.-M. Nicaud, Institute National de la Recherche Agronomique, UMR Chimie
Biologique, Thiverval Grignon, France
D. Kretzschmar, Oregon Health and Sciences University, Portland, Oregon, USA
Publications
1. Athenstaedt, K.
Neutral lipids in yeast: synthesis, storage and degradation.
In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N.
Timmis) Springer-Heidelberg, 2010, 471-480
2. Athenstaedt, K.
Players in the neutral lipid game – proteins involved in neutral lipid metabolism in
yeast.
In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N.
Timmis) Springer-Heidelberg, 2010, 537-546
3. Athenstaedt, K.
Isolation and characterization of lipid particles of yeast.
In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N.
Timmis) Springer-Heidelberg, 2010, 4223-4229
4. Athenstaedt, K.
Neutral lipid metabolism in yeast as a template for biomedical research.
In Microbiology of Hydrocarbons, Oils, Lipids, and Derived Compounds. (Ed. by K. N.
Timmis) Springer-Heidelberg, 2010, 3381-3382
26

Biophysical chemistry group
Group leader: Albin Hermetter
Post-docs: Heidrun Susani-Etzerodt, Jo-Anne Rasmussen, Heidemarie Ehammer
Graduate students: Maria Morak, Maximilian Schicher, Ute Stemmer, Daniel Koller, Claudia
Ramprecht, Lingaraju Marlingapla Halasiddappa
Diploma student: Bojana Stojcic
Technicians: Elfriede Zenzmaier
General description
Our research deals with the role of glycero(phospho)lipids and lipid modifying enzymes as
components of membranes and lipoproteins, their function as mediators in cellular
(patho)biochemistry, and their application as analytical tools in enzyme technology.
Fluorescence spectroscopy is used as a main technique to investigate the behaviour of these
biomolecules in the respective supramolecular systems.
Section 1 of the following report summarizes our studies on lipid oxidation, the effects of
oxidized lipids on intracellular signalling, and the inhibition of these processes by synthetic
and natural antioxidants.
Section 2 describes the development of fluorescence and chip technology for functional
proteomic analysis of lipolytic enzymes in microbial, animal and human cells.
1. Lipid oxidation and atherosclerosis – Lipotoxicity
1.1. Interaction of oxidized phospholipids with vascular cells
Sphingomyelin
acid
Sphingomyelinase
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
27
OXPHOS-EuroMEMBRANE
Interactions of oxidized lipoproteins with
the cells of the arterial wall induce and
influence the progress of atherosclerosis.
Accumulation of foam cells originating
from macrophages and excessive intimal
growth of vascular smooth muscle cells
(SMC) alternating with focal massive cell
death are characteristics typical of the atherosclerotic lesion. These phenomena are largely
mediated by the oxidized phospholipid components of the modified particles that are
generated under the conditions of oxidative stress. In the framework of the research consortia
LIPOTOX (SFB) and OXPHOS (EuroMEMBRANE), we investigate the “Toxicity of
oxidized phospholipids in macrophages” and “The protein targets and apoptotic signaling of
oxidized phospholipids”. These studies aim at identifying the molecular and cellular effects of

an important subfamily of the oxidized phospholipids containing long hydrocarbon chains in
position sn-1 and short polar acyl residues in position sn-2 of the glycerol backbone. The
respective compounds trigger an intracellular signaling network including sphingomyelinases,
(MAP) kinases and transcription factors thereby inducing proliferation or apoptosis of
vascular cells. Both phenomena largely depend on the specific action of sphingolipid
mediators that are acutely formed upon cell stimulation by the oxidized lipids. Fluorescence
microscopic studies on labeled lipid analogs revealed that the short-chain
phosphatidylcholines are easily transferred from the aqueous phase into the cell plasma
membrane and eventually spread throughout the cells. Under these circumstances, the biologi-
cally active compounds are very likely to interfere
directly with various signaling components inside
the cells, which finally decide about cell growth or
death. Investigations are being performed on the
levels of the lipidome, the apparent enzyme
activities, the proteome an the transcriptome to find
the primary molecular targets and their downstream
elements that are the key compnents of lipid-induced
cell death.
1.2. Oxidative stress and antioxidants
Antioxidants protect biomolecules against
oxidative stress and thus, may prevent its
pathological consequences. Specific fluorescent
markers have been established for high-throughput
screening of lipid and protein oxidation and its
inhibition by natural and synthetic antioxidants.
These methods can also be used for the
determination of antioxidant capacities of
biological fluids (e.g. serum) and edible oils. A
prominent example is pumpkin seed oil which
contains a variety of antioxidants (e.g. tocopherols
and phenolic substances) and other secondary plant
components that possess useful biological
properties.
2. Functional proteomic analysis of lipolytic enzymes
The functional properties of lipases and phospholipases are the subject of our studies in the
framework of two joint research programs (GOLD-Genomics of Lipid-associated Disorders at
KFU Graz and the Research center Applied Biocatalysis at TU Graz). Lipolytic enzymes
catalyze intra- and extracellular lipid degradation. Dysfunctions in lipid metabolism may lead
to various diseases including obesity, diabetes or atherosclerosis. In chemistry, lipases and
28

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:
Reacti v e p h osphonate gr o up,
irreversibly inhibits the catalytic serine
BG:
Binding group: is specifically recognized
by the active enzyme
NBD : fluorescent tag λ : 488 nm; λ : 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
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.
2.2. Protein microarrays – Enzyme chips
In the framework of the Kplus Research Centre
Applied Biocatalysis (TU Graz), we have
developed "biochips" representing ordered
microarrays of biospecific ligands for the detection
and quantification of lipolytic enzymes in
biological samples. In addition, microarrays of
enzymes are generated to determine protein
affinities for substrate analogs. The development
of these tools basically requires three steps, namely
specific loading of activated supports with
bioaffinity ligands, measurement of lipase binding
(e.g. by fluorescence techniques), and application
30
Angewandte
Biokatalyse
Kompetenzzentrum GmbH
of the bio chips to the investigation of real analytical problems. These systems can be used
for the screening of lipases with specific substrate acceptance.
Diploma thesis completed:
Bojana Stojcic: Fluorescence imaging of oxidized phospholipid uptake into cultured
macrophages.
Plasma lipoproteins are oxidized as a consequence of oxidative stress (enzymatic and
nonenzymatic), which in the end can cause lipid and protein modifications of the particles.
Lipid modifications can induce generation of oxidized phospholipids (oxPL) primarily from
(poly)unsaturated diacyl- and alk(en)ylacyl glycerophospholipids including
palmitoyloxovaleroyl-PC (POVPC) and palmitoylglutaroyl-PC (PGPC). Fluorescent analogs
of POVPC and PGPC labeled with BODIPY (POVPE-BY and PGPE-BY, respectively) were
used as tools for studying uptake and intracellular stability of these compounds in cultured
macrophages. The aim of this study was to understand in more detail how oxPL are delivered
to the cells and to analyze their metabolic fate inside the cells. The emphasis was on the role
of the (supra)molecular carriers delivering oxPL to the cells including lipid micelles, albumin
and (mm)LDL. The import of the fluorescent lipids from these donors into macrophages was
studied using fluorescence microscopy. In addition, we analyzed the transfer of oxPL between
albumin and LDL and the degradation products of PGPE-BY as well as the complexes of the
chemically reactive POVPE-BY with proteins or lipids. From our results, it can be inferred
that POVPC mainly elicits its biological effects as a conjugate with lipids and/or proteins. In
order to understand the biological activites of PGPC, we have to take into account that they
are mediated by various compounds including the intact oxPL and its metabolites..

Doctoral Theses completed:
Maria Morak: Functional proteomic analysis and comparison of lipolytic enzymes in mouse
tissues
Lipases and esterases catalyze the degradation of carboxylic acid esters including mono-, di-,
and triacylglycerols, and cholesteryl esters. The hydrolysis of these substrates is a key process
in energy homeostasis, lipid remodeling and signaling of eucaryotes. Disorders of lipid
homeostasis may lead to obesity, type 2 diabetes, atherosclerosis, dyslipidaemia and other
lipid-associated diseases. In order to identify the active lipolytic enzymes in complex
proteomes of mouse tissues, fluorescent and biotinylated p-nitrophenyl phosphonic acid esters
were used as activity-recognition probes for selective protein tagging followed by
electrophoretic separation and analysis by nanoHPLC-MS/MS. In addition, differential
activity-based gel electrophoresis (DABGE) was developed as a novel technique for the
comparison of lipolytic enzymes from two different biological samples in one gel. This
method was applied to compare the lipolytic proteomes of brown and white adipose tissue
from wild-type mice and animals deficient in the key enzymes responsible for intracellular
TAG hydrolysis in these organs. We found characteristic lipase patterns in BAT and WAT
that specifically responded to the expression of ATGL (adipose triglyceride lipase) and HSL
(hormone-sensitive lipase).
Maximilian Schicher: Functional proteomic analysis of lipolytic enzymes in cultured murine
and human fat cells
Lipolytic enzymes are responsible for intra- and extracellular degradation of acylglycerols and
cholesterol esters. The hydrolysis of these compounds is a key event in energy homeostasis
and signaling processes in mammals and humans. Impairment of lipid metabolism may lead
to diseases such as insulin resistance, obesity, type 2 diabetes, atherosclerosis and other lipidassociated
disorders. We used a functional proteomics approach based on fluorescent and
biotinylated p-nitrophenyl phosphonates as activity-based probes to identify active lipolytic
and esterolytic enzymes in subcutaneous and visceral human adipocytes as well as
undifferentiated and differentiated murine fat cells. The labeled proteins were separated by gel
electrophoresis, imaged with a laser scanner and identified by LC-MS/MS after in-gel
digestion. Furthermore, we compared the lipolytic activities of subcutaneous and visceral
adipocytes in a single polyacrylamide gel by differential activity-based gel electrophoresis.
We found specific enzyme patterns depending on the differentiation state and localization of
fat cells. The functional analysis of overexpressed proteins revealed novel lipase candidates.
International Cooperations:
T. Hianik, Department of Biophysics and Chemical Physics, Comenius University, Bratislava,
Slovakia
T. Hornemann, Institute for Clinical Chemistry, University Hospital Zürich; Zürich,
Switzerland
M. Hof, Department of Biophysical Chemistry, Academy of Sciences of the Czech Republic,
Prague
M. Palmer, Department of Chemistry, University of Waterloo, Ontario, Canada
D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine;
Cornell University, Ithaca, USA
31

R. Yates, Department of Biochemistry and Molecular Biology, Faculty of Medicine,
University of Calgary, Calgary, Canada,
N. Faergeman, Department of Biochemistry and Molecular Biology, University of Southern
Denmark, Odense, Denmark
Research Projects:
BM.W_F Genomforschung in Österreich (GEN-AU): Exploring the lipolytic proteome.
Phospholipases (GOLD 3 project-Genomics of Lipid-associated Disorders, coordinated
by KFU Graz).
FWF Special Research Program (SFB) LIPOTOX (coordinated by KFU Graz): Toxicity of
oxidized phospholipids in macrophages.
FWF Doctoral Program Molecular Enzymology: Functional enzyme analysis.
FWF EUROCORES-EuroMEMBRANE-OXPHOS-Protein targets and apoptotic signaling of
oxidized phospholipids
TIG Research center Applied Biocatalysis (coordinated by TU Graz): Novel lipases with
specific substrate acceptance.
Invited lectures:
1. Signaling of oxidized phospholipids in vascular cells. Fluorescent inhibitors for
functional proteomic analysis of lipases. Biocrates, Innsbruck, January 21, 2009.
2. Toxicity of oxidized phospholipids in macrophages. COST meeting “Free radicals in
Chemical Biology”, Gniezno, Poland, September 11, 2009.
3. The lipolytic proteomes of mouse adipose tissue and liver. Cornell University, Ithaca,
NY, USA, September 22, 2009
4. Fluorescent inhibitors for functional proteomic analysis of lipolytic enzymes. Euro Fed
Lipid, Graz, Austria, October 20, 2009
Publications:
1. Rhode S, Grurl R, Brameshuber M, Hermetter A, Schütz GJ
Plasma membrane fluidity affects transient immobilization of oxidized phospholipids in
endocytotic sites for subsequent uptake.
J. Biol. Chem. 284, 2258-65, 2009
2. Watschinger K, Keller MA, Hermetter A, Golderer G, Werner-Felmayer G, Werner ER
Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal
ion and tetrahydrobiopterin dependence.
Biol. Chem. 390, 3-10, 2009
3. Yates RM, Hermetter A, Russell DG
Recording phagosome maturation through the real-time spectrofluorometric
32

measurement of hydrolytic activities.
Methods Mol. Biol. 531, 157 -171, 2009
4. Schicher M, Kollroser M, Hermetter A
Mapping the lipolytic proteome of adipose tissue using fluorescent suicide inhibitors
Methods Mol. Biol. 579, 497-511, 2009.
5. Morak M, Schmidinger H, Krempl P, Rechberger G, Kollroser M, Birner-Gruenberger
R, Hermetter A
Differential activity-based gel electrophoresis for comparative analysis of lipolytic and
esterolytic activities.
J. Lipid Res. 50, 1281-92, 2009.
6. Landre JB, Blaess MF, Kohl M, Schlicksbier T, Ruryk A, Kinscherf R, Claus RA,
Hermetter A, Keller M, Bauer M, Deigner HP
Addressable bipartite molecular hook (ABMH): Immobilized hairpin probes with
sensitivity below 50 femtomolar
Anal. Biochem. 2009, Epub ahead of print.
33

Chemistry of Functional Foods
Group leader: Michael Murkovic
Graduate students: Alam Zeb, Yuliana Reni Swasti
Diploma students: Katrin Julia Greimel, Thomas Binder, Irma Sliumpaite, Bahar Nahimi
Zahabi, Ramune Bobinaite
Technician: Alma Ljubijankic
Honorary Professor: Klaus Günther, Forschungszentrum Jülich, Germany
Project Assistant: Sonja Thanner-Lechner
General description
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 foods 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 foods 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 foods and many of them are highly toxic and/or carcinogenic.
However, these hazardous compounds occur in rather low concentrations being normally not
acute toxic. The substances have a very diverse chemical background like heterocyclic
amines, polycondensated aromatic compounds, acrylamide or furan derivatives. The aim of
the research is to investigate the reaction mechanisms that lead to the formation of these
hazardous compounds and establish strategies to mitigate the formation and thereby reducing
the alimentary exposure.
Carotenoids thermal oxidation in triacylglycerols
β-Carotene is one of most important and widespread carotenoids present in food. It is an
important source of provitamin A. It acts as antioxidant in biological systems including the
complex system of lipids. We studied the oxidation of β-carotene in model triacylglycerols
system, comparable to most of the high oleic edible oil. An HPLC electrospray ionization
mass spectrometric method was developed for the separation and identification of
triacylglycerols and its oxidation products (Figure 1). β-Carotene was found to act as
antioxidant at lower temperature like 90 and 100 o C, while oxidation of triacyglycerols was
closely correlated with the oxidation of β-carotene after 8 hours of thermal oxidation at
110 o C. The oxidized products of β-carotene were epoxides, peroxides and apocarotenals,
separated and analyzed using C30 reversed phase HPLC-DAD and APCI-MS. We found that
110 °C and 120 o C were the favorable temperatures for studying the oxidation reaction of
34

carotenoids, while 110 o C, 120 o C and 130 o C were favorable for triacylglycerol oxidation.
Stability of triacylglycerols decrease with the increased formation of the oxidation products of
β-carotene.
Intensity
100
80
60
40
20
Trilinolein
288
263
O
O
O
O
+ [ M + Na]
901
[ M + NH 4]
+
896
+ [ M + H]
879
0
200 300 400 500 600 700 800 900
O
O
m/z
LL +
599
DAGs
Intensity
1-Oleoyl-2-linoleoyl-3-linoleoyl glycerol
100
O
80
60
40
20
Intensity
1-Oleoyl-2-linoleoyl-3-oleoyl glycerol
100
80
60
40
20
+
[ M-
O + H + Li]
874
288
263
+ [ M + H]
883
0
200 300 400 500 600 700 800 900
O
O
O
LL
599
+
+ [ M + Na]
903
[ M + NH4]
+
898
+
[ M-
O + H+
Li]
288
+ 872
[ M + H]
263
881
0
200 300 400 500 600 700 800 900
O
m/z
O
OL
601
+
O
O
O
O
m/z
O
O
OL +
601
OO
603
+
0 5 10 15 20 25 30
Time (min)
35
[ M + Na]
+ 905
[ M + NH4]
+
900
Intensity
4
603
[ M + NH ] +
100 OO 902
+
O
Triolein
80
60
40
O
O
O
20
+
[ M-
O + H + Li]
876
+ [ M + H]
263
883
0
200 300 400 500 600 700 800 900
O
O
m/z
Intensity
1-Oleoyl-2-oleoyl-3-stearoyl glycerol
100
O
80
60
40
20
[ M + Na]
+
907
O
O
O
O
OO
603
+
OS
605
+
[ M + NH4]
+
909
[ M + Na]
+
904
288
+
[ M + H]
887
0
200 300 400 500 600 700 800 900
O
HPLC-MS
Figure 1. Separation and Identification of Triacylglycerols using HPLC-ESI-MS.
Diploma Theses completed
Thomas Binder: Effects of blanching and enzyme treatment on the amount of asparagine in
potato slices
The overall aim of this work was to find different ways to reduce the asparagine content in
raw potato slices. This was done by studying the effect of blanching and enzyme treatments
on the asparagine content in raw and blanched potato slices. A further important objective was
to find a good treatment for lowering the asparagine content in potato slices which also can be
used in the food industry. For this reason the cooking value was calculated for the different
blanching treatments.
Katrin Julia Greimel: Absolute quantification of soy in animal feed
The quantification of technically unavoidable contaminations in animal feed (especially soy)
is necessary due to the threshold values determined by law. However, it turns out to be a great
challenge for analytical laboratories. In this work, two different methods for the absolute
quantification of the soy percentage in animal feed have been developed. One method for
detection is based on molecular biology the real-time polymerase chain reaction (real-time
PCR), the other is a standard method of classical analytical chemistry, namely the high
performance liquid chromatography (HPLC). Sensitive and specific primers were developed
for the PCR-method and were additionally used with suitable hydrolysis probes in a
multiplex-PCR. The optimisation of the method made it possible to quantify low percentage
soy in animal feed products. The quantification by HPLC was carried out by means of the two
isoflavones daidzein and genistein, that are especially present in soybeans. Both were chosen

ecause they are not present in other feed ingredients. Using acid hydrolysis for the isolation
of the isoflavones, it was possible to determine the amount of soy in different animal feeds.
The focus of this work was set on the comparison of the two methods in terms of
reproducibility and accuracy.
International Cooperations
R. Venskutonis, Institute of Food Technology, Kaunas University of Technology, Lithuania
T. Husoy, National Institute of Public Health, Olso, Norway
H.R. Glatt, Deutsches Institute für Ernährungsforschung, Potsdam Rehbrücke, Germany
E. Lazos, TEJ of Athens, Greece
R. Grujic, University of East Sarajevo, Bosnia and Herzegovina
E. Habul, University of Sarajevo, Bosnia and Herzegovina
E. Winkelhausen, S. Kuzmanova, University Ss Cyril and Methodius, Skopie, FRYM
H. Pinheiro, Instituto Superior Tecnico, Lisboa, Portugal
V. Piironen, Department of Applied Chemistry and Microbiology, Helsinki, Finland
M.J. Cantalejo, Department of Food Technol., Public University of Navarre, Pamplona, Spain
Research Projects
European Network of Excellence: EuroFIR European Food Information Resource
Invited Lectures
1) M. Murkovic
Alimentary Antioxidants: Relevance in Foods and some Nutritional Aspects
Seminar at JRC-IHCP. 10 Dec. 2009
2) M. Murkovic
Formation of hazardous compounds during heating of food
Thermally processed foods: possible health implications - Analytical and chemical
aspects related to thermally processed foods. Aveiro, 17 Apr. 2009
Publications
1) Nugroho Prasetyo, E., Kudanga, T., Steiner, W., Murkovic, M., Nyanhongo, G. S.,
Gübitz, G.
Antioxidant activity assay based on laccase-generated radicals
Analytical and bioanalytical chemistry 393 (2009) 2, 679 - 687
2) Murkovic, M.
Europäische Lebensmitteldatenbank: European Food Information Resource Journal für
Ernährungsmedizin 11 (2009) 3/4, 28 – 29
3) Pricelius, S., Murkovic, M., Souter, P., Gübitz, G.
Substrate specificities of glycosidases from Aspergillus species pectinase preparations
on elderberry anthocyanins
Journal of agricultural and food chemistry 57 (2009) 3, 1006 - 1012
36

Lectures and Laboratory Courses
Anleitung zu wissenschaftlichen Arbeiten aus Biochemie 2 SE Daum G, Hermetter A,
und Molekularer Biomedizin
Macheroux P
Anleitung zu wissenschaftlichen Arbeiten aus Biochemie 2 SE Daum G, Hermetter A,
und Molekularer Biomedizin
Macheroux P
Bachelorarbeit 1 SE Daum G
Bachelorarbeit 1 SE Daum G
Bioanalytik 2.25 VO Hermetter A, Klimant I
Biochemie I 3,75 VO Macheroux P
Biochemie I LU 5,33 LU Staff
Biochemie II 1,5 VO Macheroux P
Biochemie II LU 4 LU Staff
Biophysikalische Chemie 3 PV Laggner P
Biophysikalische Chemie 3 PV Laggner P
Biophysikalische Chemie der Lipide 3 PV Hermetter A
Biophysikalische Chemie der Lipide 3 PV Hermetter A
Chemie und Biowissenschaften 4 VU Hermetter A,
Breinbauer R, Gübitz
G
Chemie und Technologie der Lebensmittel II 2 VO Murkovic M
Chemische Veränderungen in Lebensmitteln 2 PV Murkovic M
Chemische Veränderungen in Lebensmitteln bei der
Verarbeitung
2 SE Murkovic M
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
GL Chemie (BMT) 2 VO Hermetter A
Immunologische Methoden 2 VO Daum G
Immunologische Methoden 2 LU Daum G
Immunologische Methoden 1 VO Daum G
Lebensmittelbiotechnologie 1,3 VO Murkovic M
Lebensmittelbiotechnologie 6 PR Murkovic M
Lebensmittelchemie/Technologie 4 VU Murkovic M
Lebensmittelchemie/Technologie 3 VU Murkovic M
Lebensmitteltechnologisches Praktikum 4 LU Murkovic M
Lebensmitteltechnologisches Praktikum 1 SE Murkovic M
Membran Biophysik 3 PV Lohner K
Membran Biophysik 3 PV Lohner K
Membran-Mimetik 1 VO Lohner K
Molekularbiologie LU 3 LU Waldner-Scott I, Knaus
T
Molekulare Enzymologie 2 VO Gruber K, Macheroux
P, Nidetzky B
Molekulare Enzymologie I 3 PV Macheroux P
37

Molekulare Enzymologie II 3 PV Macheroux P
Molekulare Gastronomie und Lebensmittelverarbeitung II 1 EV Murkovic M
Molekulare Physiologie 2 VO Macheroux P
Physik.Meth.i.d.Biochemie 2 LU Laggner P
Physikalische Methoden in der Biochemie 1 VO Laggner P
Projektlabor Bioanalytik 6 PR Daum G, Hermetter A,
Macheroux P
Projektlabor Bioanalytik 6 PR Daum G, Hermetter A,
Macheroux P
Qualitätssicherung in Pharma-, Lebensmittel- und
Biotechnologie
2 VO Murkovic M
Seminar zu den LU aus Molekularbiologie 1 SE Athenstaedt K,
Waldner-Scott I
Seminar zur Masterarbeit aus Biochemie und Molekularer 2 SE Daum G, Hermetter A,
Biomedizin
Macheroux P
Seminar zur Masterarbeit aus Biochemie und Molekularer 2 SE Daum G, Hermetter A,
Biomedizin
Macheroux P
Spezielle Kapitel der Biochemie 1 VO Daum G, Hermetter A,
Macheroux P
Spezielle Kapitel der Lebensmittelchem.- u. technologie I 2 SE Murkovic M
Spezielle Kapitel der Lebensmittelchemie- und techn.II 2 SE Murkovic M
Strukturanalyse in Biophysik u. Materialforschung 2 VO Laggner P
Umwelt- und Lebensmittelbiotechnologie 4 LU Murkovic M
Warenkunde - Lebensmittel 1 SE Murkovic M
Zellbiologie 1 1 SE Daum G
Zellbiologie 2 1 SE Daum G
Zellbiologie der Lipide 3 PV Daum G
Zellbiologie der Lipide 3 PV Daum G
38