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

Graz University of Technology
Austria
Institute of Biochemistry
Annual Report 2012
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Staff Members of the Institute of Biochemistry, TU Graz
http://www.biochemistry.tugraz.at/
Professors
Peter Macheroux (Full Professor & Head of the Institute)
(peter.macheroux@tugraz.at; Tel.: +43-(0)316-873-6450)
Günther Daum (Associate Professor)
(guenther.daum@tugraz.at; Tel.: +43-(0)316-873-6462)
Albin Hermetter (Associate Professor)
(albin.hermetter@tugraz.at; Tel.: +43-(0)316-873-6457)
Michael Murkovic (Associate Professor)
(michael.murkovic@tugraz.at; Tel.: +43-(0)316-873-6495)
Assistants
Dr. Alexandra Binter
(alexandra.binter@tugraz.at; Tel.: +43-(0)316-873-6453)
Dr. Ines Waldner-Scott
(ines.waldner-scott@tugraz.at; Tel.:+43-(0)316-873-4522)
DI Tanja Knaus
(tanja.knaus@tugraz.at; Tel.: +43-(0)316-873-6463)
Dr. 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
Alma Ljubijankic; alma.ljubijankic@tugraz.at; Tel.: +43 (316) 873 - 6460, 6498
Sabrina Moratti, sabrina.moratti@tugraz.at; Tel.: +43(0)316-873-4522
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 2012
Peter Macheroux
Following his nomination for the “Excellence in teaching” award at
Graz University of Technology, Peter Macheroux received the
“Alumni prize for excellent teaching” sponsored by Dr. Odorich
Susani in February 2012. As a reflection of the successful NAWI
Graz master program “Biochemistry and molecular Biomedicine”,
six master students completed their master thesis in 2012. In
addition, the group was also very successful in publishing in high
caliber journals such as PNAS and Angewandte Chemie (see list of
publications). Both publications resulted from collaborative efforts
within the FWF-funded PhD program in “Molecular Enzymology”
(with Prof. Karl Gruber and Prof. Rolf Breinbauer, respectively)
and emphasize the added value of the program and its “open
laboratory” policy.
Research in the group of Albin Hermetter led to the discovery that
oxidized phospholipids display preferential cytotoxicity in
(skin)cancer cells (PhD thesis, C. Ramprecht). Poster prizes were
awarded for this work to C. Ramprecht at the 8 th Doc Day of NAWI
Graz and the Annual ÖGBMT Meeting in Graz. An international
(PCT) patent application has been filed by TU Graz to cover this
invention. A. Hermetter has been appointed member of the journal
Methods and Applications in Fluorescence. He is also a permanent
member of the international steering committee of a conference
series devoted to the same topic. Together with P. Kinnunen/
Finland and C.M. Spickett/ UK, he served as Guest Editor of a
special issue for Biochim. Biophys. Acta/Biomembranes, which is
devoted to Oxidized phospholipids-their properties and interactions
with proteins.
Claudia Ramprecht
Susanne Horvath
The laboratory of Günther Daum continued research in the fields
of yeast organelles, biomembranes and lipids. The most successful
work of PhD students was internationally well recognized and
appreciated. Susanne E. Horvath received the JBC/Herbert Tabor
Young Investigator Award at the 53rd International Conference on
the Bioscience of Lipids (ICBL)/ASBMB Symposium, Banff,
Canada; and Birgit Ploier received the Poster Award at the GSA
Yeast Genetics and Molecular Biology meeting, Princeton
University, USA. Martina Gsell had the opportunity of a 6 months
stay at Amyris Biotechnologies Inc. Company, Emeryville, CA,
USA. As research related activities, Günther Daum continued his
work as a Board Member of the Austrian Science Fund (FWF), as
an Executive Editor of Progress in Lipid Research and as Associate
Editor of FEMS Yeast Research.
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In the group Chemistry of Functional Foods directed by Michael
Murkovic in 2012 a PhD thesis was finalized in which the
polymerization of furfuryl alcohol was investigated. Furfuryl alcohol
is formed during heating of foods, e.g. during roasting of coffee. It
can be activated metabolically which might result in the induction of
tumors. The polymerization removes the furfuryl alcohol from the
foods and reduces the bioavailability. In 2012 several international
students (Lithuania, Hungary, Slovak Republic, Thailand, Indonesia)
were coming to the lab for carrying out specific experiments related
to, e.g. new technologies of heating, oxidation of oils in the bulk
phase and in emulsions, and the application of asparaginase for
reduction of the acrylamide formation, determination of antioxidants,
characterization of bioactive peptides isolated from fish waste.
Michael Murkovic
Edina Harsay
In 2012, Edina Harsay from the Proteogenomics Research Institute
for Systems Medicine in San Diego, CA, USA, spent a semester in
our Institute as a Guest Professor of NaWi Graz and Fulbright
Fellow. Edina Harsay who has previously been Assistant Professor
at the Department of Molecular Biosciences, University of Kansas,
Lawrence, USA, is interested intracellular membrane trafficking,
cellular stress response, cell growth and stress signaling pathways.
During her stay in Graz she was involved in teaching activities of
our institute and also visited research institutions in Austria and
other European countries.
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Biochemistry Group
Group leader: Peter Macheroux
Secretary: Annemarie Portschy
Senior scientists: Alexandra Binter, Ines Waldner, Silvia Wallner
PhD students: Peter Augustin, Thomas Bergner, Bastian Daniel, Venugopal Gudipati, Tanja
Knaus, Karin Koch, Wolf-Dieter Lienhart, Chaitanya Tabib, Chanakan Tongsook
Master students: Altijana Hromic, Karin Koch, Julia Koop, Katharina Lukas, Stefanie
Monschein, Barbara Steiner, Nicole Sudi
Technicians: Sabrina Moratti, Eva Maria Pointner, Rosemarie Trenker-El-Toukhy
General description
The fundamental questions in the study of enzymes, the bio-catalysts of all living organisms,
revolve around their ability to select a substrate (substrate specificity) and subject this
substrate to a predetermined chemical reaction (reaction and regio-specificity). In general,
only a few amino acid residues in the "active site" of an enzyme are involved in this process
and hence provide the key to the processes taking place during enzyme catalysis. Therefore,
the focus of our research is to achieve a deeper understanding of the functional role of amino
acids in the active site of enzymes with regard to substrate-recognition and stereo- and
regiospecificity of the chemical transformation. In addition, we are also interested in
substrate-triggered conformational changes and how enzymes utilize cofactors (flavin,
nicotinamide) to achieve catalysis. Towards these aims we employ a multidisciplinary
approach encompassing kinetic, thermodynamic, spectroscopic and structural techniques. In
addition, we use site-directed mutagenesis to generate mutant enzymes to probe their
functional role in the mentioned processes. Furthermore, we collaborate with our partners in
academia and industry to develop inhibitors for enzymes, which can yield important new
insights into enzyme mechanisms and can be useful as potential lead compounds in the design
of new drugs.
The methods established in our laboratory comprise kinetic (stopped-flow and rapid quench
analysis of enzymatic reactions), thermodynamic (isothermal titration microcalorimetry) and
spectroscopic (fluorescence, circular dichroism and UV/VIS absorbance) methods. We also
frequently use MALDI-TOF and ESI mass spectrometry, protein purification techniques
(chromatography and electrophoresis) and modern molecular biology methods to clone and
express genes of interest. A brief description of our current research projects is given below.
Berberine bridge enzyme & other flavin-dependent oxidases in the model plant
Arabidopsis thaliana
Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a
pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD
moiety, which is essential for catalysis. The reaction involves the oxidation of the N-methyl
group of the substrate (S)-reticuline by the enzyme-bound flavin and concomitant formation
of a carbon-carbon bond (the “berberine bridge”). The ultimate acceptor of the substratederived
electrons is dioxygen, which reoxidizes the flavin to its resting state:
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The BBE-catalysed oxidative carbon-carbon bond formation is a new example of the
versatility of the flavin cofactor in biochemical reactions. Our goal is to understand the
oxidative cyclization reaction by a biochemical and structural approach.
We have developed a new expression system for BBE (using cDNA from Eschscholzia
california, gold poppy) in Pichia pastoris, which produces large amounts of the protein (ca.
500 mg from a 10-L culture). The availability of suitable quantities of BBE enabled us to
crystallize the protein and to solve the structure in collaboration with Prof. Karl Gruber at the
University of Graz (see below).
Based on the three-dimensional structure of BBE, we have performed a site-directed
mutagenesis program to investigate the role of amino acids present in the active site of the
enzyme. In conjunction with other experiments, this has led to the formulation of a new
reaction mechanism for the enzyme (thesis project of Andreas Winkler). Currently, we study
the functional role of several amino acid residues interacting with the isoalloxazine ring of the
flavin cofactor in order to get a deeper understanding of the reaction and to enable the
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expression of more versatile biocatalysts. Together with the structural biology group at the
University of Graz we focus on oxygen reactivity of BBE and the role of certain amino acids
in this process (master project of Altijana Hromic and Barbara Steiner).
Using bioinformatics we searched for BBE homologs in sequence databases and found these
enzymes to be widespread among plants, fungi and bacteria. We started to characterize BBEhomologs
from A. thaliana, where we focus on their role in metabolism and on covalent
modifications of the embedded flavin. We investigate whether alternative covalent
modifications of the flavin are feasible, i.e. whether other nucleophilic amino acid side chains
(e.g. aspartate or tyrosine) can form a covalent bond to the 8-methylgroup or to the 6
position of the isoalloxazine ring. A BBE homolog was identified in A. thaliana bearing a
histidine in close proximity to the 6 position of the isoalloxazine ring making a new covalent
modification feasible. This BBE-homolog was expressed in P. pastoris and the crystal
structure of the protein was solved in collaboration with Prof. Karl Gruber (see below: left,
overall topology; right, active site of AtBBE). The crystal structure showed a monocovalent
linkage of the flavin cofactor. Further studies will focus on an in-depth biochemical
characterization of the enzyme and on the determination of natural substrates in order to
understand the role of this protein in planta (thesis project of Bastian Daniel)
Luciferase and LuxF
The emission of light by biological species (bioluminescence) is a fascinating process found
in diverse organisms such as bacteria, fungi, insects, fish, limpets and nematodes. In all cases
the bioluminescent process is based on a chemiluminescent reaction in which the chemical
energy is (partially) transformed into light energy ("cold light"). All bioluminescent processes
require a luciferase, i.e. an enzyme catalyzing the chemiluminescent reaction, and a luciferin,
which can be considered a coenzyme. During the bioluminescent reaction the luciferin is
generated in an excited state and serves as the emitter of light energy. In our laboratory, we
are interested in the bioluminescence of marine photobacteria. In these bacteria, luciferase is
composed of an alpha/beta-heterodimeric protein, which binds reduced flavinmononucleotide
(FMN) as the luciferin. The reduced FMN reacts with molecular dioxygen to a hydroperoxide
intermediate with subsequent oxidation of a long-chain fatty aldehyde (e.g. tetradecanal) to
the corresponding fatty acid (e.g. myristic acid). During this oxidation process, an excited
flavin intermediate is generated which emits light. Some marine photobacteria possess an
additional protein called LuxF which was found in complex with a myristylated flavin
derivative where the C-3 atom of myristic acid is covalently attached to the 6-position of the
flavin ring system. It was postulated that this flavin adduct is generated in the luciferase
catalyzed bioluminescent reaction. Furthermore, it was speculated that LuxF sequesters the
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myristylated flavin adduct in order to prevent inhibition of the bioluminescent reaction.
However, both hypotheses have not been tested on a biochemical or physiological level yet.
Hence, in this study we will design and perform experiments to examine the putative
generation of myristylated FMN through the luciferase reaction (thesis project of Thomas
Bergner and Chaitanya Tabib)
Structure of a LuxF dimer in the
absence (red) and presence (blue)
of the myristylated flavin
derivative (pdb code 1NFP)
Lot6p – a redox regulated switch of the proteasome
During our previous studies of bacterial quinone reductases, we have also investigated the
biochemical properties of the yeast homolog Lot6p. Despite the availability of a threedimensional
structure for Lot6p (1T0I), the physiological role of the enzyme was unclear.
Our recent studies have now demonstrated that the enzyme rapidly reduces quinones at the
expense of a reduced nicotinamide cofactor, either NADH or NADPH. In order to further
characterize the cellular role of Lot6p, we have carried out pull-down assays and identified
the 20S core particle of the yeast proteasome as interaction partner. Further studies revealed
that this complex recruits Yap4p, a member of the b-Zip transcription factor family, but only
when the flavin-cofactor of Lot6p is in its reduced state. Oxidation of the flavin leads to
dissociation of the transcription factor and relocalization to the nucleus. Our current efforts
focus on how Lot6p exerts its regulatory role on Yap4p and the function of this transcription
factor in gene expression (thesis project of Venugopal Gudipati and Karin Koch).
A similar system is known from mammalian cells, where a homologous quinone
reductase (NQO1) binds to the 20S proteasome and recruits important tumor suppressor
proteins such as p53 and p73. Interestingly, the NQO1 gene is polymorphic and the two
most common variants in humans feature single amino acid replacements (P187S and R???W,
termed variant 1 and 2, respectively). Since variant 1 was implicated in tumorigenesis, we are
currently characterizing the recombinant protein structurally and biochemically to explain its
role in this medically important process (thesis project of Wolf-Dieter Lienhart).
Reverse structural flavogenomics
Several structures of new flavoproteins with unknown biological function have been
elucidated by structural genomics initiatives. For example, in a zinc-dependent putative
protease from Bacteroides thetaiotaomicron the flavin isoalloxazine ring is sandwiched by
two tryptophans at the interface of the dimeric protein (pdb entry 3CNE, see model below):
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This unusual binding mode and the large distance to the presumed catalytic zinc center argue
against a catalytic role of the bound flavin. A detailed characterization of the flavin binding
site using natural and chemically modified flavin derivatives showed that binding is solely
governed by the aromatic -stacking interaction and does not involve the N(10)-side chain of
the flavin. In support of this finding, replacement of the tryptophan by phenylalanine gave rise
to much weaker binding, whereas in the tryptophan to alanine variant, flavin binding was
abolished. Therefore, we propose that the protein is an unspecific scavenger of flavin
compounds and may serve as a storage protein in vivo. Current efforts focus on the
characterization of the zinc binding site and its potential role as a catalytic center (thesis
project of Tanja Knaus and master project of Julia Koop and Stefanie Monschein).
Similarly, the structure of an uncharacterized flavoprotein exhibiting a pyruvate kinase C-
terminal domain-like fold was elucidated by the Joint Center for Structural Genomics (pdb
code 1VP8, model see below):
This protein constitutes a novel flavin binding pocket and appears to be specific to archeons
such as Archeoglobus fulgidus and Methanobacterium thermoautotrophicum. We have
expressed the protein from A. fulgidus in Escherichia coli and are now in the process to
characterize it in order to unravel its biochemical function (joint project of Bastian Daniel and
Chanakan Tongsook).
Doctoral theses completed
Silvia Wallner: Flavoproteins with bicovalent flavin tethering
Flavoproteins are a large and diverse group of proteins that possess a non-, mono- or
bicovalently bound FMN or FAD cofactor for catalysis. Covalent attachment of the cofactor
occurs when either the 8alpha- or 6-position of the isoalloxazine forms a covalent bond to the
amino acid side chain of a cysteine, histidine or tyrosine residue, respectively. Flavoproteins
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with bicovalent cofactor linkage are a novel class of proteins which are present in both
prokaryotic and eukaryotic organisms. These enzymes have extraordinary properties, such as
unusually high redox potentials that enable catalysis of challenging chemical reactions in
plants, fungi and bacteria as was shown by redox potential determinations. Moreover,
bicovalent linkage appears to be crucial for structural integrity of the protein and correct
positioning of the flavin in the active site. Many bicovalently-linked flavoenzymes can be
exploited as capable biocatalysts; hence, a detailed understanding of these enzymes is of
utmost importance. This work is intended to get a deeper insight in the role of bicovalent
flavin tethering and to identify and characterize new members of this class of proteins. (S)-
Tetrahydroprotoberberine oxidase (STOX) was identified as a new bicovalent flavoprotein in
poppy and berberis species and was found to be involved in benzylisoquinoline alkaloid
biosynthesis by catalyzing the stereoselective oxidation of various (S)-
tetrahydroprotoberberines. In the course of this thesis project a system for heterologous
protein expression was established using Pichia pastoris as host organism, which resulted in
the formation of low quantities of STOX and in the identification of (S)-tetrahydropalmatine
as preferred substrate of STOX from Argemone mexicana. A further objective was to perform
in-depth studies on berberine bridge enzyme (BBE), a paradigm for bicovalent flavoproteins
which is used for the biocatalytic production of (S)-berbines and (R)-benzylisoquinolines. We
created BBE variants, which should accept non-natural substrates leading to the formation of
new pharmacologically active compounds. Moreover, biochemical studies on BBE showed
that His174 seems to stabilize the uptake of negative charge into the isoalloxazine ring system
during catalysis. The respective variant protein features a reduced midpoint potential of the
cofactor and significantly decreased overall enzyme efficiency and hence provides further
insights in the catalytic properties of BBE.
Tanja Knaus: Biochemical characterization and mechanistic studies on two zinc dependent
proteins
Pisa1 from Pseudomonas sp. DSM6611: A highly enantio- and stereoselective secondary
alkylsulfatase from Pseudomonas sp. DSM6611 (Pisa1) was heterologously expressed in
Escherichia coli BL21 and purified to homogeneity for kinetic and structural studies.
Structure determination of Pisa1 by X-ray crystallography showed that the protein belongs to
the family of metallo-ß-lactamases with a conserved binuclear Zn2+ cluster in the active site.
In contrast to a closely related alkylsulfatase from Pseudomonas aeruginosa (SdsA1), Pisa1
showed preference for secondary rather than primary alkyl sulfates and enantioselectively
hydrolyzed the (R) enantiomer of rac-2-octyl sulfate yielding (S)-2-octanol with inversion of
absolute configuration as a result of C-O bond cleavage. In order to elucidate the mechanism
of inverting sulfate ester hydrolysis, for which no counterpart in chemical catalysis exists, an
invariant histidine residue (His317) near the sulfate binding site was identified as the general
acid for crucial protonation of the sulfate leaving group. Additionally, amino acid
replacements in the alkyl-chain binding pocket generated an enzyme variant, which lost its
stereoselectivity towards rac-2-octyl sulfate. These findings are discussed in light of the
potential use of this enzyme family for applications in biocatalysis. ppBat from Bacteroides
thetaiotaomicron: In a recent survey of flavin-dependent proteins, a putative protease from
Bacteroides thetaiotaomicron, a microbe inhabiting the human gut, was identified. The
structure of the protein (pdb code 3CNE) features a mononuclear zinc site in addition to a
flavin, which is sandwiched by two tryptophan residues provided by each of the two
protomers in the dimeric protein. The zinc ion is coordinated with two cysteine-derived thiol
groups and two water molecules forming a tetragonal ligation sphere. The large distance of ≈
16 Å between the edge of the flavin cofactor and the zinc argues against a direct cooperation
between these two cofactors. This intriguing combination of unusual cofactors sparked our
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interest in the protein. In this thesis, the biochemical characterization of the flavin as well as
the zinc binding site of the protein is reported. Moreover it is demonstrated, that the
recombinant protein is capable of binding not only naturally occurring flavin derivatives, such
as lumichrome, riboflavin, FMN and FAD but also a variety of chemically modified
“artificial” flavin analogs with K D values in the nanomolar range. Additionally, it could be
proved that the Trp164 is fundamental for binding the flavin cofactors but not for a
dimerization of the protein and that the zinc ion has a structural role in ppBat.
Master theses completed
Altijana Hromic: Characterisation of His174Asn variant of berberine bridge enzyme from
Eschscholzia californica
Flavoproteins are a large group of proteins that use FAD or FMN for catalysis. In most
flavoproteins this cofactor is non-covalently bound, however both monocovalent and
bicovalent attachment modes are also known. Bicovalent flavinylation gives the respective
enzymes a lot of interesting properties, such as a high redox potential. Moreover, bicovalent
linkage can be crucial for holding the flavin cofactor in the active site and for structural
integrity. Berberine bridge enzyme (BBE) belongs to the class of bicovalently flavinylated
oxidases. It is a fundamental enzyme of alkaloid biosynthesis in specific plant families like
Berberidaceae or Papaveraceae. BBE catalyses the oxidative ring closure of the N-methyl
group of (S)-reticuline to the C8 atom of (S)-scoulerine. One important active site residue in
BBE is His174. It was identified as crucial because of its role in stabilisation of the reduced
state of the flavin cofactor. In this work I present the biochemical characterisation of a BBE
His174Asn variant. From sequence alignments it is known that His174 of BBE is strictly
conserved among bicovalently linked flavoprotein oxidases and that an asparagine residue is
present at the respective position of bicovalently flavinylated dehydrogenases (like Phl p 4
and TrdL). Biochemical parameters like redox potential and turnover rates were determined
and compared with wild type BBE and BBE His174Ala. An approximately seven-fold
decrease in catalytic activity and a two-fold decrease in redox potential was determined for
His174Asn in comparison with BBE wild type. A possible reason for this decrease is absence
of a hydrogen bond network between His174 and the isoalloxazine ring system of flavin
cofactor when His174 is replaced with asparagine. Therefore, a crystal structure of the
His174Asn variant would be necessary to prove this hypothesis.
Karin Koch: Cellular role of the yeast bZip transcription factor Yap4p
The yeast bZip-transcription factor Yap4p belongs to the Yap-family, which comprises eight
members. Most family-members are involved in stress response programs. In order to fulfill
this function the expression is regulated by cis-regulatory elements and posttranslational
modification of the transcription factors. Furthermore the subcellular localization and
degradation of the proteins provides regulation mechanisms. For example Yap4p, interacts
with the quinone reductase Lot6p. Lot6p plays a crucial role in regulating ubiquitin
independent degradation of Yap4p by the 20S proteasome. Furthermore, localization of
Yap4p is affected by the Lot6p-20S proteasome complex. Under oxidative stress Yap4p is
released from the complex and translocates into the nucleus were it affects the expression of
its target genes. Previous attempts to express and purify recombinant hexa-histidine-tagged
Yap4p in Escherichia coli were not successful in the desirable purity and amount. Therefore
the main aim of this work was to obtain recombinant nona-histidine-tagged Yap4p using
Saccharomyces cerevisiae as host strain in a suitable form so that it can be used for further
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characterization. Additionally the effect of overexpression of recombinant Yap4p under
normal and under different stress conditions was studied. Expression and purification attempts
of recombinant nonahistidine-tagged Yap4p were not successful. However, overexpression of
recombinant nonahistidine-tagged Yap4p led to a delay in the cell cycle in the G1 phase and
changed the pattern of lipid droplet formation. Yap4p overexpression also led to significantly
increased cell survival after oxidative stress exposure. This indicated that Yap4p could have
greater influences in oxidative stress response than assumed and should be more thoroughly
investigated.
Julia Koop: Characterization of flavin binding properties of the putative protease I from
Bacteroides thetaiotaomicron and isolation of flavin from bovine milk
B. thetaiotaomicron is a gram-negative microbe found in the human gut system and is
frequently associated with serious wound infections, peritonitis and septicaemia. Recently, a
putative hydrolase with unprecedented binding properties was discovered in this
microorganism. The occurrence of a binding site for flavin derivatives in addition to a
mononuclear zinc and a calcium binding site is uncommon for hydrolases. Based on structural
relationships this enzyme was classified as a putative protease. A special feature of the flavin
binding site is its unspecific binding of different forms of flavins (e. g. riboflavin, FMN, FAD
and chemically modified flavins). Not only naturally occurring, but also chemically modified
flavins bind with high affinity between the two tryptophanes of the site. Strength and
preferences of cofactor binding to the protein were analyzed by UV/vis absorbance
spectroscopy. The successful binding of flavins to the protein gave rise to a facilitated method
of cofactor isolation from various biological sources, such as milk, urea and blood, as well as
an improved technique for detection of flavins. Different bovine and human milk samples
were used for isolation of riboflavin, FMN and FAD with the recombinant apo-protein. All
experiments showed riboflavin as the major compound from various milk samples.
Furthermore, a distinction between free and protein-bound riboflavin could be achieved. Only
a small amount of riboflavin occurred in a free state, the majority was present in a proteinbound
form, which was obtained after precipitating the milk proteins with 100% trichloro
acetic acid. However, the isolated amounts of riboflavin from the milk samples varied
distinctly indicating a high heterogeneity of the composition of milk.
Katharina Lukas: Recombinant expression of 2-aminobenzoyl-CoA monooxygenase /
reductase (ACMR) of Azoarcus evansii
2-aminobenzoyl-CoA monooxygenase/ reductase (ACMR) is a bifunctional flavoenzyme
from A. evansii, consisting of two functional domains, a 2-aminobenzoyl-CoA
monooxygenase (ACM) and a 2-aminobenzoyl-CoA reductase (ACR). This organism uses a
complex mechanism to degrade aromatic compounds, such as 2-aminobenzoate. ACMR is
one of the essential enzymes in this metabolism, which catalyses the redox reaction of 2-
aminobenzoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA, a nonaromatic
product. The main focus of this work was to remove the spacer region between ACM and
ACR in order to express them separately. Furthermore it was important to determine the
required cofactors, which bind to ACMR during this redox reaction. Therefore, flavins, such
as FAD or FMN play an essential role. Results show that FAD binds to the ACM domain to
act as an electron donor for molecular oxygen. The same applies to FMN, which binds to the
ACR domain, to act as a prosthetic group. Moreover recombinant expression was performed
to obtain more details about the structure and function of this enzyme and the domains,
respectively. For that reason the proteins were expressed separately, brought into a soluble,
pure form to be available for further protein crystallisation and eventually structure
determination by X-ray analysis.
14

Stefanie Monschein: Functional characterization of a putative protease from Bacteroides
thetaiotaomicron (ppBat)
After structural determination of a putative protease of B. thetaiotaomicron, a flavin- and an
atypical zinc bind site were discovered. In this study the two cysteines, forming the zinc
binding site, were replaced by alanine in single and double replacements. Due to the strong
binding of the wild-type to riboflavin, difference titrations and ITC measurements of the
variants were conducted. The binding affinity properties of ppBat variants to riboflavin were
detected as high as the properties of the wild type. Differences between the variants and the
wild type occurred in protein stability, tested by CD and Thermofluor® measurements. The
altered zinc binding site had a negative influence on the temperature sensitivity of the protein.
Possible restructuring of the second structure of the protein to more α-helical content is
proposed. The putative protease activity of ppBat, a member of the DJ-1 superfamily, could
not be detected by gelatin and casein zymography. Chaperone activity, present in other
members of this superfamily, should be tested in further experiments for clear conclusions.
Nicole Sudi: Generation of mikkomycin in fram deletion mutants in Streptomyces tendae
Tü901/8c
Nikkomycins belong to the group of nucleoside peptidyl antibiotics produced by several
Streptomyces species. They show fungicidal, insecticidal and acaricidal properties due to the
inhibition of chitin synthase, which is liable for the formation of a protective cell wall in
various fungi, insects and other species. Because of this function, nikkomycins are promising
compounds in the cure of immunosuppressed patients. Furthermore, nikkomycins might be
important new antibiotics in the therapy of several fungal diseases like blastomycosis,
candidiasis or coccidioidomycosis, for which nikkomycin Z is in clinical trials. They are
comprised of a peptidyl and a nucleoside moiety. The biosynthesis of the peptidyl moiety,
which is called nikkomycin D or hydroxypyridylhomothreonine (HPHT), is very well
investigated. The biosynthesis of the nucleoside moiety, also called aminohexuronic acid
moiety, however, is almost unknown. The initial step of this biosynthetic pathway is catalyzed
by the enzyme NikO, which is responsible for the formation of 3’-enolpyruvyl-UMP by its
enolpyruvyl transferase function. With the gene nikO, further genes are encoded on the same
operon: nikI, nikJ, nikK, nikL, and nikM. The enzymes encoded on this operon might be
involved in the biosynthesis of the aminohexuronic acid moiety. Based on sequence
similarities to known proteins putative functions were assigned to some of the encoded
enzymes. In order to investigate the role of these enzymes more precisely, a PCR-targeting
system was used to generate in frame deletion mutants of S. tendae Tü901/8c, a highly
effective nikkomycin producing strain. For this work, the knockouts were made using a
cosmid, by replacing the genes with a resistance marker, which was then excised by using a
recombinase system. After preparing the mutated cosmids in Escherichia coli, they were
transformed into the nikkomycin producing strain, in which the genes were deleted in the
process of homologous recombination.
International cooperations
Maria Abramic, Ruder Boskovic Institute Zagreb, Croatia
Steve Ealick, Cornell University, Ithaca, U.S.A.
Toni Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A.
Shwu Liaw, National Yang-Ming University, Taipei, Taiwan
Matthias Mack, Hochschule Mannheim, Germany
15

Research projects
FWF P24189: “Bacterial bioluminescence”
FWF P22361: “Mechanism of redox controlled protein degradation”
FWF-Doktoratskolleg “Molecular Enzymology”
WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49
family”
Invited Lecture
1) Reverse structural flavogenomics: Discovery of function of uncharacterized
flavoproteins. Trends in Enzymology, 5. June 2012, Göttingen, Germany
Publications
1) Bezerra, G. A., Dobrovetsky, E., Viertlmayr, R., Dong, A., Binter, A., Abramic, A.,
Macheroux, P., Dhe-Paganon, S., and Gruber, K.: Entropy driven binding of opioid
peptides induces a large domain motion in human DPPIII, Proc. Natl. Acad Sci. U.S.A.,
2012, 109:6525-6530.
2) Schober, M., Knaus, T., Tösch, M., Macheroux, P., Wagner, U., Faber, K.: The
substrate spectrum of the inverting sec-alkylsulfatase Pisa1, Adv. Synth. Catal., 2012,
354:1737-1742.
3) Krysiak, J. M., Kreuzer, J., Macheroux, P., Hermetter, A., Sieber, S. A., and Breinbauer,
R.: Activity-based probes for studying the activity of flavin-dependent oxidases and for
the protein target profiling of monoamine oxidase inhibitors, Angew. Chem. Int. Ed.,
2012, 51:7035-7040; Angew. Chem., 2012, 124:7142-7147.
4) Wallner, S., Winkler, A., Riedl, S., Dully, C., Horvath, S., Gruber, K., Macheroux, P.:
Catalytic and structural role of an active site histidine 174 in berberine bridge enzyme,
Biochemistry, 2012, 51:6139-6147.
5) Knaus, T., Eger, E., Koop, J., Stipsits, S., Kinsland, C., Ealick, S. E., Macheroux, P.:
Reverse structural genomics: An unusual flavin binding site in a putative protease from
Bacteroides thetaiotaomicron, J. Biol. Chem., 2012, 287:27490-27498.
6) Durchschein, K., Wallner, S., Macheroux, P., Schwab, W., Winkler, T., Kreis, W.,
Faber, K.: Nicotinamide-dependent ene-reductases as alternative biocatalysts for the
reduction of activated alkenes, Eur. J. Org. Chem., 2012, 4963-4968.
7) Oberdorfer, G., Binter, A., Ginj, C., Macheroux, P., Gruber, K.: Structural and
functional characterization of NikO, an enolpyruvyl transferase essential in nikkomycin
biosynthesis, J. Biol. Chem., 2012, 287:31427-31436.
8) Resch, V., Lechner, H., Schrittwieser, J. H., Wallner, S., Gruber, K., Macheroux, P.,
Kroutil, W.: Inverting the regioselectivity of berberine bridge enzyme employing
customized fluorine-containing substrates, Chemistry, 2012, 18:13173-13179.
16

9) Durchschein, K., Wallner, S., Macheroux, P., Zangger, K., Fabian, W. M. F., Faber, K.:
Unusual C=C-bond isomerization of an ,-unsaturated -butyrolactone catalyzed by
flavoproteins from the Old Yellow Enzyme family, ChemBioChem, 2012, 13:2346-
2351.
10) Knaus, T., Schober, M., Kepplinger, B., Faccinelli, M., Pitzer, J., Faber, K., Macheroux,
P., Wagner, U.: Structure and mechanism of the first alkylsulfatase specific for
secondary alkylsulfates, FEBS J., 2012, 279:4374-4384.
11) Jajcanin-Jozic, N., Macheroux, P., Abramic, M.: Yeast ortholog of peptidase family
M49: The role of Gul461 and Tyr327, Croatica Chemica Acta, 2012, 85:535-540.
12) Wallner, S., Dully, C., Daniel, B., Macheroux, P.: Berberine bridge enzyme and the
family of bicovalent flavoenzymes, in “Handbook of Flavoproteins” (Vol. 1), Hill, R.,
Miller, S., Palfey, B., eds., Walter de Gruyter, Berlin, 2012, p. 1-30.
17

Cell Biology Group
Group leader: Günther Daum
Postdoctoral Fellow: Karlheinz Grillitsch (ACIB)
PhD students: Susanne Horvath, Martina Gsell, Vid V. Flis, Vasyl’ Ivashov, Lisa Klug,
Claudia Schmidt, Barbara Koch, Birgit Ploier
Master students: Stefanie Horvath, Martina Zandl
Technicians: Claudia Hrastnik (half time), Alma Ljubijankic (half time)
Guest Professor: Edina Harsay, NaWi Graz Fulbright Fellow; Proteogenomics Research
Institute for Systems Medicine, San Diego, CA, USA)
General description
Lipids are important cellular components. Depending on the cell type they can serve as
storage molecules which are mobilized under conditions of energy requirements. In all types
of cells lipid, especially phospholipids and sterols, are important building blocks of cellular
membranes.
Our laboratory has a long standing tradition to study biogenesis and maintenance of biological
membranes and assembly of lipids into organelle membranes using the yeast as an
experimental model system. This cellular model system has become a valuable tool to
investigate principles of cell biology but also to address specific questions concerning the
distinct function of various components. We combine biochemical, molecular and cell
biological methods addressing problems of lipid metabolism, lipid depot formation and
membrane biogenesis.
Specific aspects studied recently in our laboratory are (i) biosynthesis and assembly of
phosphatidylethanolamine (PE) in yeast organelles with emphasis on the role of PE in
mitochondria; (ii) non-polar lipid metabolism in the yeast and regulatory aspect affecting
formation and mobilization of lipid depots; and (iii) isolation and characterization of organelle
membranes from the yeast Pichia pastoris in connection to lipid metabolism.
Phosphatidylethanolamine, a key component of yeast organelle membranes
Phosphatidylethanolamine (PE) is one of the major phospholipids of yeast membranes. It is
highly important for membrane stability and integrity and thus also for cell function and
proliferation. PE synthesis in the yeast is accomplished by four different pathways, namely
(i) synthesis of phosphatidylserine (PS) in the endoplasmic reticulum and decarboxylation by
mitochondrial phosphatidylserine decarboxylase 1 (Psd1p); (ii) synthesis of PS and
conversion to PE by the Golgi localized Psd2p, (iii) the CDP-ethanolamine pathway
(Kennedy pathway) in the endoplasmic reticulum, and (iv) the lysophospholipid acylation
route catalyzed by Ale1p and Tgl3p. To obtain more insight into biosynthesis, assembly and
homeostasis of PE, single and multiple yeast mutants bearing defects in the respective
pathways are required.
18

Fig. 1: Pathways of phosphatidylethanolamine formation in the yeast
Previous investigations in our laboratory were aimed at the molecular biological
identification of novel components involved in PE homeostasis of the yeast Saccharomyces
cerevisiae. For this purpose, genetic screenings and DNA microarray analysis were
performed which led to the discovery of a number of candidate genes. In particular, 54 yeast
genes were significantly up-regulated in the absence of PSD1 compared to wild type.
Surprisingly, marked down-regulation of genes was not observed. A number of different
cellular processes in different subcellular compartments were affected in a ∆psd1 mutant.
Deletion mutants bearing defect in all 54 candidate genes, respectively, were analyzed for
their growth phenotype and their phospholipid profile. Only three mutants, namely ∆gpm2,
∆gph1, ∆rsb1, were affected in one of these parameters. The possible link of these mutations
to PE deficiency and PSD1 deletion was anticipated. Especially the contribution of Gph1p,
which was originally identified as a glycogen phosphorylase required for the mobilization of
glycogen, turned out to be of interest. Biochemical and cell biological investigations are in
progress to pinpoint the additional molecular role of Gph1p in yeast lipid metabolism.
PE homeostasis in the yeast cell is linked to traffic of this phospholipid between various
compartments. Currently, the link between PE metabolism and peroxisome proliferation is
subject of investigation with emphasis on the role of enzymes and lipid transport routes
involved. Previous studies had suggested that PE formed through all four pathways (see
above) in different subcellular membranes can be supplied to peroxisomes with comparable
efficiency. However, mechanisms involved in these translocation processes are still unclear.
Using various mutants and employing cell biological methods such as the use of
permeabilized yeast cells PE transport to peroxisomes can be studied. The contribution of the
different PE biosynthetic pathways to the supply of PE to peroxisomes and mechanisms of
PE translocation between organelles are central aspects of this study. Recently, these
investigations were extended to the supply of phosphatidylcholine (PC) to peroxisomes. Also
in this case the emphasis is on the contribution of different PC forming pathways (PE
methylation and CDP-choline pathway) and modes of PC translocation to this organelle.
The major player in phosphatidylethanolamine synthesis in the yeast is the mitochondrial
phosphatidylserine decarboxylase 1 (Psd1p). Like most mitochondrial proteins, Psd1p is
synthesized on free cytosolic ribosomes and imported into mitochondria where processing
occurs. The Psd1-proenzyme contains a mitochondrial targeting sequence, an internal sorting
sequence, and an alpha- and a beta-subunit which are linked through an LGST cleavage site.
19

Cleavage at this site leads to the mature and active form of the enzyme generating a pyruvoyl
group at the N-terminus of the alpha subunit.
Fig. 2: Psd1 domain structure displaying the predicted transmembrane domains IM1 and
IM2, the substrate recognition site (SRS), the LGST motif and the N-terminal targeting
sequences (MT).
In a most fruitful collaboration with the laboratory of Prof. N. Pfanner and colleagues,
Freiburg, Germany, we investigated (i) the precise import route of Psd1p through
mitochondrial membranes; (ii) the specific role of the LGST cleavage site on the import,
assembly and maturation of the enzyme;, (iii) the topology of Psd1p in the inner
mitochondrial membrane; (iv) the effect of mitochondrial processing peptidases on protein
maturation; and (v) possible complex formation of mature Psd1p.
Within the 46 kDa β-subunit, the two possible transmembrane domains IM1 and IM2 were
predicted to anchor the enzyme to the inner mitochondrial membrane. To get more
information about the mechanism(s) targeting Psd1p to the inner mitochondrial membrane/
intermembrane space we constructed a series of Psd1p variants lacking IM1, IM2 or both
transmembrane domains. Moreover, we deleted a domain within the α-subunit which is
predicted to be the substrate recognition site. These Psd1p variants were analyzed for
processing and maturation, their phospholipid composition as well as their PS decarboxylase
activity. All three domains of Psd1p were shown to affect import and assembly of the protein
into mitochondria as well as the enzymatic activity. We conclude that IM1 serves as a
membrane anchor for the β-subunit, whereas IM2 targets the enzyme to the inner
mitochondrial membrane. We also hypothesize that the potential substrate recognition site
within the α-subunit is not only involved in recognition of PS, but also in Psd1p processing
and stability.
Deletion of PSD1 causes PE depletion in mitochondria which affects protein complexes of
the outer and inner mitochondrial membrane. In a series of experiments, we observed an
impaired function of the translocase of the outer membrane (TOM complex), decrease of the
inner membrane potential affecting the import of preproteins into and across the inner
membrane and reduced respiratory capacity, whereas the formation of larger respiratory
chain supercomplexes was initiated. Finally, we investigated whether alterations of the
mitochondrial morphology in MINOS (inner membrane organizing system) mutants are
related to the mitochondrial lipid status. Lack of MINOS core components did not affect the
mitochondrial phospholipid composition. Thus, we excluded that defects observed in these
mutants are secondary effects caused by changes in the mitochondrial lipid profile.
20

Fig. 3: Interaction of PE with outer and inner mitochondrial protein complexes. PE affects the
import of β-barrel precursor proteins at the stage of the TOM complex, decreases the inner
membrane potential (Δψ), the import efficiency in and across the inner mitochondrial
membrane (TIM23 and TIM22 complexes) and the cytochrome c oxidase activity (IV). PE
favors the formation of larger respiratory chain supercomplexes between cytochrome bc1 (III)
and cytochrome c oxidase (IV). AAC, ADP/ATP carrier; IMM, inner mitochondrial
membrane; OMM, outer mitochondrial membrane; PE, phosphatidylethanolamine; SAM,
sorting and assembly machinery.
Storage of non-polar lipids in lipid droplets and mobilization
Yeast cells like most other cell types have the capacity to store non-polar lipids. In the case of
Saccharomyces cerevisiae triacylglycerols (TG) and SE (steryl esters) are the predominant
lipid storage molecules which accumulate in subcellular structures named lipid particles/
droplets. Upon requirement, TG and SE can be mobilized and serve as building blocks for
membrane biosynthesis. In a long-standing project of our laboratory, we investigate the
characterization of enzymatic steps which lead to formation and mobilization of TG and SE
depots.
Previous studies in our laboratory had identified Tgl3p, Tgl4p and Tgl5p as the major yeast
TG lipases. Surprising evidence was obtained when the enzymology of the three TG lipases
Tgl3p, Tgl4p and Tgl5p was studied. Motif search analysis indicated that Tgl3p, Tgl4p and
Tgl5p do not only contain the TG lipase but also acyltransferase motifs. Indeed, all three
enzymes exhibit lipase activity but also catalyze acylation of lysophosphatidylethanolamine
and lysophosphatidic acid, respectively. In addition to Tgl3p, Tgl4p and Tgl5p a number of
candidate gene products with potential lipase/esterase activity were also identified. Some of
these candidate gene products were identified by the conserved GXSXG-type lipase motif,
others also by their localization to the lipid droplets were the substrate for the lipase reaction,
TG is stored.
Currently, molecular properties of the three yeast TG lipases including their topology on lipid
droplets and the functional link between non-polar lipid formation and degradation are
studied. Balance between formation and degradation of TG and SE is important because
excess of fatty acids in cells may cause a lipotoxic effect, whereas fatty acid depletion may
21

esult in structural or regulatory defects. Recently, we showed that catabolic and anabolic
reactions channeling fatty acids from storage TG to phospholipids are linked to multiple
functions of the TG lipases Tgl3p, Tgl4p and Tgl5p which do not only act as lipolytic
enzymes but also contribute to lipid synthesis through their function as acyltransferases. Lack
of the TG synthesizing enzymes Dga1p and Lro1p affects the activity of Tgl3p, Tgl4p and
Tgl5p and vice versa.
Fig. 4: Life cycle of yeast non-polar lipids and genes involved in non-polar lipid formation
and degradation.
Topology of TG lipases on the surface of lipid droplets and interaction with other components
of the lipid biosynthetic machinery are important issues to understand the physiological role
of these enzymes in more detail. To address this problem we established a protocol for limited
proteolysis of lipid droplet surface proteins and focused first on the orientation of N- and C-
terminus of Tgl3p. The C-terminus of this lipase seems to be deeply embedded in the lipid
droplet which may be related to targeting and anchoring of Tgl3p to this organelle. However,
Tgl3p is not exclusively located to lipid droplets but is also present in the endoplasmic
reticulum (ER). This finding raises the question how the same protein can be assembled into a
monolayer (lipid droplets) and a bilayer membrane (ER). The specific interaction of Tgl3p
with its membrane environment and/or specific partner proteins may be a clue to this problem.
Pichia pastoris organelles and lipids
The yeast Pichia pastoris is an important experimental system for heterologous expression of
proteins. Surprisingly, little is known about organelles of this microorganism. For this reason,
we started a systematic biochemical and cell biological study to establish standardized
methods of Pichia pastoris organelle isolation and characterization. Recent work focused on
the biochemical characterization of the plasma membrane and secretory organelles from
Pichia pastoris. Moreover, lipid droplets from Pichia pastoris were isolated and analyzed
with respect to their lipids and proteins.
22

Transmission electron micrographs
of Pichia pastoris wild type cells
grown on glucose (YPD) and oleate
(YPO). WT: wild type; TM:
dga1∆lro1∆are2∆ triple mutant
Pictures shown be courtesy of Dr. G.
Zellnig, University of Graz, Austria
In Pichia pastoris like in many other eukaryotes, fatty acids are stored in the biologically inert
form of triacylglycerols (TG) and steryl esters (SE) as energy reserve and/or as membrane
building blocks. Recently, we identified gene products catalyzing formation of TG and SE in
this yeast. Based on sequence homologies to Saccharomyces cerevisiae, the two
diacylglycerol acyltransferases Dga1p and Lro1p and one acyl CoA:sterol acyltransferase
Are2p from Pichia pastoris were identified. Mutants bearing single and multiple deletions of
the respective genes were analyzed for their growth phenotype, their lipid composition and
their ability to form lipid droplets. Our results indicate that the above mentioned gene
products are most likely responsible for the entire TG and SE synthesis in Pichia pastoris.
Lro1p appears to be the major TG synthase in this yeast, whereas Dga1p contributes less to
TG synthesis. In contrast to Saccharomyces cerevisiae, Are2p is the only SE synthase in
Pichia pastoris. Interestingly, TG formation in Pichia pastoris is indispensable for lipid
droplet biogenesis. The small amount of SE synthesized by Are2p in a dga1∆lro1∆ double
deletion mutant is insufficient to initiate the formation of the storage organelle.
Besides lipid droplets, the endoplasmic reticulum from Pichia pastoris gained our special
interest. This organelle is not only the major biosynthetic site of proteins and lipids within the
cell, but also the starting point for protein secretion. An isolation protocol for microsomes
(subfractions of the endoplasmic reticulum) was established and fractions obtained were
analyzed for their proteome and lipidome. Identification of protein and lipid components of
the endoplasmic reticulum is important because the molecular equipment of this subcellular
compartment is subject to modifications caused by environmental changes.
Master Thesis completed
Stefanie Horvath: Molecular Characterization of Phosphatidylserine Decarboxylase 1 from
Yeast
In yeast, phosphatidylserine decarboxylase 1 (Psd1) is the major enzyme catalyzing the
formation of phosphatidylethanolamine (PE) from phosphatidylserine (PS). Psd1 is composed
23

of an α- and a β-subunit and located to the intermembrane space and the inner mitochondrial
membrane, respectively. Within the 46 kDa β-subunit, two possible transmembrane domains
IM1 and IM2 were predicted to anchor the enzyme to the inner mitochondrial membrane. To
get more information about the mechanism(s) targeting Psd1 to the inner mitochondrial
membrane/intermembrane space we constructed a series of Psd1 variants lacking IM1, IM2 or
both transmembrane domains. Moreover, we deleted a domain within the α-subunit which is
predicted to be the substrate recognition site in analogy to mammalian Psd. For detailed
molecular characterization of Psd1 processing/maturation, mitochondria of the respective
mutant strains were isolated and analyzed. Phospholipid analysis and enzyme assays revealed
a requirement for IM2 not only for Psd1 function and processing, but also for correct targeting
to the inner mitochondrial membrane. Although deletion of IM1 does not inhibit cleavage of
Psd1 into α- and β-subunits, a mislocalization of both non- identical subunits to the matrix
side of the inner mitochondrial membrane was observed. We conclude that IM1 serves as a
membrane anchor for the β-subunit, whereas IM2 targets the enzyme to the inner
mitochondrial membrane. Moreover, IM2 may also contribute to Psd1 maturation by
formation of α- and β-subunits. We also hypothesize that the potential substrate recognition
site within the α-subunit is not only involved in recognition of phosphatidylserine, but also in
Psd1 processing and stability.
International cooperations
N. Pfanner, Institute of Biochemistry and Molecular Biology, ZBMZ, University of Freiburg,
Germany
M. Karas, Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University,
Frankfurt, Germany
Research projects
FWF P21429: Phosphatidylserine decarboxylase
FWF P23029 Lipases of the yeast Saccharomyces cerevisiae
FWF TRP 009 Pichia Lipidomics (Translational Research)
FWF PhD Program: Molecular Enzymology
Austrian Center of Industrial Biotechnology (ACIB): Pichia pastoris Cell factory and Protein
Production
Invited Lectures
1) G. Daum
Pichia pastoris: Black Box or Law and Order
Pichia 2012, Alpbach, Austria, 29 February – 3 March 2012
2) G. Daum, C. Schmidt, B. Koch, B. Ploier and K. Athenstaedt
Life cycle of non-polar lipids in the yeast Saccharomyces cerevisiae.
Biocatalysis in Lipid Modification. Greifswald, Germany: 19 – 21 September 2012
3) G. Daum, C. Schmidt, B. Koch, B. Ploier and K Athenstaedt
Endoplasmic reticulum and lipid droplets of the yeast interact during synthesis, storage
24

and mobilization of non-polar lipids.
EMBO Conference Series: The Physiology of the Endoplasmic Reticulum (ER):
Function and Dysfunction.
Girona, Spain, 15-19 October 2012
4) G. Daum, C. Schmidt, B. Koch, B. Ploier and K Athenstaedt
Life cycle of non-polar lipids in the yeast Saccharomyces cerevisiae
Biotechnology of Cellular Membranes (ACIB Educational Workshop)
Graz, Austria, 6-7 December, 2012
Publications
1) Spanova, M., Zweytick, D, Lohner, K., Klug, L., Leitner, E., Hermetter, A. and Daum,
G. Influence of squalene on lipid particle/droplet and membrane organization in the
yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1812 (2012) 647-653
2) Bohnert, M., Wenz, L.-S., Zerbes, R. M., Horvath, S. E., Stroud, D. A., von der
Malsburg, K., Müller, J. M., Oeljeklaus, S., Warscheid, B., Chacinska, A., Daum, G.,
Wiedemann, N., Becker, T., Pfanner, N. and van der Laan, M. Role of mitochondrial
inner membrane organizing system in protein biogenesis of the mitochondrial outer
membrane. Mol. Biol. Cell 23 (2012) 3948-3956
3) Böttinger. L., Horvath, S.E., Kleinschroth, T, Hunte, C., Daum, G., Pfanner, N. and
Becker, T. Phosphatidylethanolamine and Cardiolipin Differentially Affect the Stability
of Mitochondrial Respiratory Chain Supercomplexes J. Mol. Biol. 423 (2012) 677-686
4) Horvath, S. E., Böttinger, L., Vögtle, F.-N., Wiedemann, N., Meisinger, C., Becker, T.
and Daum, G. Processing and topology of the yeast mitochondrial phosphatidylserine
decarboxylase 1. J. Biol. Chem. 287 (2012) 36744-36755
5) Ivashov, V. A., Grillitsch, K., Koefeler, H., Leitner, E., Baeumlisberger, D., Karas, M.
and Daum, G. Lipidome and proteome of lipid droplets from the methylotrophic yeast
Pichia pastoris. Biochim. Biophys. Acta 1831 (2012) 282-290
6) Flis V. V. and Daum, G. Lipid transport between the endoplasmic reticulum and
mitochondria. Cold Spring Harbor Perspectives in Biology(2012) in press
7) Gsell, M. and Daum, G. Analysis of membrane lipid biogenesis pathways using yeast
genetics. Methods in Molecular Biology: Membrane Biogenesis (2012) in press
Awards
Birgit Ploier
Poster Award at the GSA Yeast Genetics and Molecular Biology meeting, Princeton
University, USA, July 31-August 5, 2012
Identification of novel hydrolytic enzymes possibly involved in non-polar lipid
metabolism of the yeast Saccharomyces cerevisiae
25

Susanne E. Horvath
JBC/Herbert Tabor Young Investigator Award at the 53rd International Conference on
the Bioscience of Lipids (ICBL)/ASBMB Symposium, Banff, Canada, 4-9 September
2012
Characterization of Phosphatidylserine Decarboxylase 1 from Yeast
Martina Gsell
Internship for a 6 months stay at Amyris Biotechnologies Inc. Company, Emeryville, CA,
USA
26

Biophysical Chemistry Group
Group leader: Albin Hermetter
PhD students: Daniel Koller, Claudia Ramprecht, Lingaraju Marlingapla Halasiddappa,
Bojana Stojcic, Franziska Vogl
Master students: Gabriel Pürstinger, Hannah Jaritz, Luise Britz, Sandra Jantscher
Technician: 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 supramolecular systems.
Section 1 of the following report summarizes our studies on the toxic effects of oxidized
phospholipids in the cells of the vascular wall and in cancer cells with emphasis on ceramide
as a mediator of lipid toxicity.
Section 2 describes the development of fluorescence methods for functional proteomic
analysis of lipolytic enzymes in animal and human cells.
1. Oxidized phospholipids and disease – Lipid toxicity
Polyunsaturated phospholipids in cell membranes and plasma lipoproteins are modified under
conditions of oxidative stress. Oxidized phospholipids (OxPL) are formed, showing a great
variety of biological activities. Many of these compounds are toxic in cells. On the one hand,
lipid toxicity may lead to pathophysiological consequences. In the cells of the arterial wall,
OxPL are involved in the initiation and progress of atherosclerosis. On the other hand, these
compounds can elicit beneficial effects. We recently found that some OxPL preferably kill
tumor cells and therefore could possess therapeutic potential in cancer treatment.
1.1. Toxicity of oxidized phospholipids in vascular cells
Interactions of oxidized lipoproteins with the cells of the arterial wall induce and influence the
progress of atherosclerosis. Accumulation of foam cells originating from macrophages and
excessive intimal growth of vascular smooth muscle cells (SMC) alternating with focal
massive cell death are characteristics typical of the atherosclerotic lesion. These phenomena
are largely mediated by the oxidized phospholipid components of the modified particles that
are generated under the conditions of oxidative stress. In the framework of the ESF project
OXPHOS (EuroMEMBRANE), we investigate the “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
27

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 microscopy studies using labeled lipid analogues revealed that the shortchain
phosphatidylcholines are easily transferred from the aqueous phase into the cell plasma
membrane and eventually spread throughout the cells. Under these circumstances, the
biologically active compounds interfere directly with signaling proteins inside the cells, that
are involved cell growth or death. Lipid toxicity is being studied on the levels of the
sphingolipidome, the enzyme activities responsible for ceramide homeostasis, and the
proteome and transcriptome to find primary molecular targets and their downstream elements
that are the functional components of lipid-induced cell death.
Sphingomyelin
acid
Sphingomyelinase
Oxidized -
Phospholipids
O
H
O
O
O
O
O
O
O
O
O
O P O
OH
O
O
O
O P O
OH
+
N
N +
Ceramide
JNK
p38 MAPK
Caspase 3
ERK
AKT/PKB
NFkB
PROLIFERATION
SURVIVAL
OXPHOS-EuroMEMBRANE
1.2. Toxicity of oxidized phospholipids in cancer cells
Oxidized phospholipids generate apoptotic
blebs in melanoma cells
Cancer is a complex disease characterized by genetic
mutations that lead to uncontrolled cell growth and
spread of abnormal cells. One in every three cancers
diagnosed is skin cancer, which is skin growth of
melanocytic and non-melanocytic cells with
differing causes and varying degrees of malignancy.
Melanomas which derive from pigment-producing
melanocytes in the basal layer of the epidermis
represent the most dangerous form of skin cancer
and although not being the most common skin
cancer type, they are responsible for most skin
cancer deaths. These tumors have a high chance of
metastasizing and becoming lethal. Surgical removal
of the tumor is only possible at a very early stage,
and chemotherapy as an overall strategy is not very
effective in treating melanomas. Only 15 % to 20 % of patients respond to chemotherapy
which typically works for less than a year and has little or no effect on survival time. In
collaboration with Dr. Schaider from the Department of Dermatology at the Medical
University of Graz, we found that oxidized phospholipids induce apoptosis in various types of
28

skin cancer cells, including melanomas and non-melanoma skin cancer. To understand the
basis of lipid-induced cell death, we studied the uptake and stability of these lipids in different
skin cancer cell lines. In addition, we analyzed apoptotic signaling pathways associated with
sphingolipid metabolism and screened for potential protein and membrane lipid targets. In
general, the toxic lipid effects were much more pronounced in cancer cells than in healthy
cells. Therefore, the results of this study can be considered a useful basis of a new generalized
approach for the therapy of skin cancer, which allows bypassing the problems due to the
genetic heterogeneity of tumor cells.
2. Functional proteomic analysis of lipolytic enzymes
The functional properties of lipases and phospholipases were the subject of our studies in the
joint research program GOLD (Genomics of Lipid-associated Disorders). These enzymes are
important for intra- and extracellular lipid degradation. Dysfunctions of lipases may be
causally related to various lipid-associated disorders including obesity, diabetes or
atherosclerosis. In chemistry, lipases and esterases are important biocatalysts for
(stereo)selective modifications of 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
Inactive
BG
RG
NBD
RG: R e a c t i v e p h o s p h o n a t e g r o u p ,
irreversibly inhibits the catalytic serine
Active
BG
BG: Binding group: is specifically recognized
by the active enzyme
Inactive
Inactive
Inhibited
RG
NBD
NBD : fluorescent tag
: 488 nm; : 540 nm
ex
em
Fluorescent inhibitors were applied to proteomic analysis of lipolytic enzymes in human and
animal cells. These studies were performed in the joint project GOLD (Genomics of Lipidassociated
Disorders/ coordinated by KFU Graz) which is a research consortium 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). The described inhibitor
29

technology enabled us to characterize the lipase patterns in human and animal tissues and
discover lipolytic enzymes with novel substrate preferences.
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, disease and lipase-targeted drugs.
Doctoral Thesis completed:
Lingaraju Marlingapla Halasiddappa: Cytotoxic effects of oxidized phospholipids in
vascular cells: Role of ceramide synthases.
Oxidized phospholipids (OxPLs), including 1-palmitoyl-2-glutaroyl-sn-glycero-3-
phosphocholine (PGPC) and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine
(POVPC) are among several biologically active derivatives that are generated during
oxidation of low-density lipoproteins (LDLs). These OxPLs are contributing factors in proatherogenic
effects of oxidized LDLs (OxLDLs), including inflammation, proliferation and
cell death in vascular cells. OxLDL also elicits formation of the lipid messenger ceramide
(Cer) which plays a pivotal role in apoptotic signaling pathways. Here we report that both
PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptotic cell death
which is also associated with increased cellular ceramide levels. Exposure of RAW 264.7
cells to POVPC and PGPC for several hours resulted in a significant increase in ceramide
synthase activity. Under the same experimental conditions, acid or neutral sphingomyelinase
activities were not affected. PGPC is more toxic than POVPC and a more potent inducer of
ceramide generation by activating a limited subset of CerS isoforms. The stimulated CerS
activities are in line with the C16-, C22-, and C24:0-Cer species that are generated under the
influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced
ceramide generation, demonstrating that OxPL-induced toxicity in macrophages is associated
30

with the accumulation of ceramide via stimulation of CerS activity. OxLDL elicits the same
cellular ceramide effects. Thus, it is concluded that PGPC and POVPC are active components
that contribute to the toxic effects of this lipoprotein.
Master Theses completed:
Gabriel Pürstinger: Toxicity of oxidized alkylacyl- and diacylglycerophosphocholines in
cultured RAW macrophages
The uptake of oxidized LDL (oxLDL) and its (phospho) lipid components by macrophages,
the formation of foam cells and apoptotic cell death are hallmarks of atherosclerosis. In
vascular smooth muscle cells (VSMC), oxPL have already been shown to be toxic [1]. It was
the aim of this study to determine effects of oxidized phospholipids (oxPL) on cell viability
and apoptosis in cultured macrophages. Specifically, the short-chain diacyl-oxPL 1 palmitoyl-
2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1 palmitoyl-2-(5-oxovaleroyl)-snglycero-3-phosphocholine
(POVPC) as well as their alkyl-acyl analogs 1-O-hexadecyl-2-
glutaroyl-sn-glycero-3-phosphocholine (E-PGPC) and 1-O-hexadecyl-2-(5-oxovaleroyl)-snglycero-3-phosphocholine
(E-POVPC) were studied. All analyzed oxPL induced apoptosis in
RAW 264.7 macrophages. PGPC and E PGPC, featuring a carboxylic group, showed similar
toxicities. However, both compounds were more toxic than their counterparts POVPC and E
POVPC possessing a reactive aldehyde function at the sn-2 position. The detailed mechanism
by which oxPL trigger apoptosis in macrophages is not fully understood. We found that acid
sphingomyelinase (aSMase) is a key signaling enzyme in this process [1]. aSMase activity
was rapidly stimulated after incubation with POVPC or E POVPC found in this study. PGPC
and E PGPC did not exhibit such an effect. Their toxicity must be due to a completely
different mechanism. There is evidence that PGPC and E PGPC induce ROS production by a
CD36 and TLR2/6 dependent pathway in other cells [2] [3]. In summary, this study showed
that oxPL are toxic to cultured macrophages. The extent of apoptosis and the pathways by
which it is induced, depend on the structure of the individual oxPL.
Luise Britz: Toxicity of oxidized phospholipids in a murine melanoma cell line.
Oxidized phospholipids are generated from (poly-) unsaturated glycerophospholipids under
conditions of oxidative stress. The truncated fatty acyl residues contain a variety of polar
functional groups which mediate the biological activity of these lipids. It was shown in
previous studies that oxidized phospholipids induce apoptosis in cells of the vascular wall and
this contributes to the development of atherosclerosis.
In this work, we provide evidence that the cytotoxicity of oxidized phospholipids can elicit
beneficial effects in inhibiting growth of malignant cells. We studied the cellular effects of the
oxidized phospholipids POVPC and PGPC as well as their ether analogues E-POVPC and E-
PGPC on the murine melanoma cell line B16-BL6. It was shown that oxidized (ether)
phospholipids induce apoptosis and necrosis in B16-BL6 mouse melanoma cells in a lipid and
concentration dependent manner. These effects were associated with efficient lipid uptake and
the formation of the apoptotic messenger ceramide. From lipidome analysis it can be inferred
that very distinct sphingolipid species are involved. This data show that oxidized
phospholipids may be considered the basis for further evaluation of their cytostatic potential
in mouse model.
31

International cooperations:
T. Futerman, Department of Biological Chemistry, Weizman Institute, Rehovot, Israel
T. Hornemann, Institute for Clinical Chemistry, University Hospital Zürich; Zürich,
Switzerland
M. Hof, Department of Biophysical Chemistry, Academy of Sciences of the Czech Republic,
Prague
I. Parmryd, Cell Biology, The Wenner-Gren Institute, Stockholm University, Stockholm,
Sweden
T. Hugel, IMETUM, TU München, Garching, Germany
P. Kinnunen, Biophysics, Aalto University, Helsinki, Finland
D. Russell, Department of Microbiology & Immunology, College of Veterinary Medicine;
Cornell University, Ithaca, USA
R.P. Kühnlein, Abteilung Molekulare Entwicklungsbiologie, Max-Planck-Institut für
Biophysikalische Chemie, Göttingen, Germany.
Research projects:
BM.W_F Genomforschung in Österreich (GEN-AU): Exploring thelipolytic proteome.
Phospholipases (GOLD 3 project-Genomics of Lipid-associated Disorders, KFU Graz).
FWF Doctoral Program Molecular Enzymology: Enzymes of ceramide signaling in response
to oxidized phospholipids.
FWF, ESF-EuroMEMBRANE-OXPHOS: Protein targets and apoptotic signaling of oxidized
phospholipids
Publications:
1) Birner-Gruenberger R, Lange J, Bickmeyer I, Hermetter A, Kollroser M, Rechberger
GN, Kühnlein RP. Functional fat body proteomics and gene targeting reveal in vivo
functions of drosophila melanogaster a-Esterase-7. Insect Biochem. Mol. Biol. 42, 220-
9, 2012
2) Watschinger K, Fuchs JE, Yarov-Yarovoy V, Keller MA, Golderer G, Hermetter A,
Werner-Felmayer G, Hulo N, Werner ER. Catalytic residues and a predicted structure of
tetrahdrobiopterin-dependent alkylglycerol monooxygenase. Biochem. J. 443, 279-86,
2012.
3) Stemmer U, Zenzmaier E, Stojcic B, Rechberger G, Kollroser M, Hermetter A. Uptake
and protein targeting of fluorescent oxidized phospholipids in cultured RAW 264.7
macrophages. Biochim. Biophys. Acta. 1821, 706-18, 2012.
4) Spanova M, Zweytick D, Lohner K, Klug L, Leitner E, Hermetter A, Daum G.
Influence of squalene on lipid particle/droplet and membrane organization in the yeast
Saccharomyces cerevisiae. Biochim. Biophys. Acta.1821, 647-53, 2012.
5) Stemmer U, Hermetter A. Protein modification by aldehydophospholipids and its
functional consequences. Biochim. Biophys. Acta 1818, 2436-45, 2012.
32

6) Keller MA, Watschinger K, Lange K, Golderer G, Werner-Felmayer G, Hermetter A,
Wanders RJA, Werner ER. Studying fatty aldehyde metabolism in living cells with
pyrene-labeled compounds. J. Lipid Res. 53, 1410-6, 2012.
7) Krysiak JM, Kreuzer J, Hermetter A, Sieber SA, Breinbauer R. Activity-based probes
for studying the activity of flavin-dependent oxidases and for the protein target profiling
of MAO-inhibitors. Angew. Chemie Int Ed Engl.51, 7035-40, 2012.
8) Stemmer U, Dunai ZA, Koller D, Pürstinger G, Zenzmaier E, Deigner, HP, Aflaki E,
Kratky D, Hermetter A. Toxicity of oxidized phospholipids in cultured macrophages.
Lipids Health Dis. 11, 110, 2012.
9) Morak M, Schmidinger H, Riesenhuber GN, Rechberger G, Kollroser M, Haemmerle
G, Zechner R, Kronenberg F, Hermetter A. ATGL and HSL deficiencies affect
expression of lipolytic activities in mouse adipose tissues. Mol. Cell. Proteomics 11,
1777-1789, 2012.
10) Oxidized phospholipids-Their properties and interactions with proteins. Special issue of
Biochim. Biophys. Acta, section Biomembranes,
P. Kinnunen, A. Hermetter, C.M. Spickett (editors), Vol. 1818, 2012.
PCT patent application :
Phospholipid compounds for use in cancer treatment.
Inventors: A. Hermetter, C. Ramprecht, H. Schaider
33

Group Chemistry of Functional Foods
Group leader: Michael Murkovic
PhD students: Yuliana Reni Swasti
Diploma students: Vanessa Verient, Wolfgang Sindler, Evelina Dimitrova, Tanja Obrietan,
Evelina Dimitrova, Natascha Fuchs, Barbara Honsig-Erlenburg
International students: Monika Ugintaite, Ausra Degutyte, György Gyany, Sureeporn
Kangsanant, Šarūnas Barnackas, Tri Wardani Widowati
Technician: Alma Ljubijankic
Honorary Professor: Klaus Günther, Forschungszentrum Jülich, Germany
Visiting Professors: Zuzana Ciesarova, Food Research Institute Bratislava, Slovakia, Chakree
Thongkraung, Prince of Songkla University, Hatyai, Thailand
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 food and their behaviour during processing and cooking is
important especially when these substances are used as food additives. The safety evaluation
of these compounds includes the evaluation of possible degradation products.
Heating of food is a process that is normally done to improve the safety and digestibility and
improve the sensory attributes like texture, colour, 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, colour, and
texture of the prepared food and many of them are highly toxic and/or carcinogenic. However,
these hazardous compounds occur in rather low concentrations being normally not acute
toxic. The substances have a very diverse chemical background like heterocyclic amines,
polycondensated aromatic compounds, acrylamide or furan derivatives. The aim of the
research is to investigate the reaction mechanisms that lead to the formation of these
hazardous compounds and establish strategies to mitigate the formation and thereby reducing
the alimentary exposure.
Polymerization of furfuryl alcohol during roasting of coffee
Analysis of furfuryl alcohol oligomers from a model system and from roasted coffee using
Ion Trap MS: The dimeric and trimeric furfuryl alcohol was also analyzed with Ion Trap MS.
From MS1 results showed that the abundant ion is m/z 161 and m/z 241 which refer to
dimeric and trimeric furfuryl, respectively. Those 2 abundant ions were then fragmented and
showed the same way of fragmentation as in LC/MS analysis as can be seen in its MS2 and
MS3. The dimeric furfuryl alcohol also found roasted coffee that was roasted at 210 °C for 4
minutes using a household roasting machine.
34

Figure 1. MS2 of dimeric of furfuryl alcohol in ion tap analysis.
Green coffee does not contain any furfuryl alcohol. However, furfuryl alcohol is formed
during roasting. After 3 min of roasting at 210 °C the formation of furfuryl alcohol starts with
a significant increase during continued roasting and reduced at 5 min roasting. This means
that a degradation of the 1,2-enediol (key intermediate in the isomerization reaction of
fructose and glucose) and the degradation of quinic acid could occur. Furfuryl alcohol
oligomers are also found after 3 min of roasting and are also reduced after 5 min of roasting.
The oligomers that were identified with LC-MS are the dimer and acid condition also occur
during coffee roasting. The dimer is present because during degradation a 1,2-enediol also is
formed under acid conditions by producing formic acid. Therefore, furfuryl alcohol is able to
polymerize under that conditions and contribute in the formation of brown colour.
Doctoral Thesis completed
Yuliana Reni Swasti: Furan Derivatives: Its Occurrence in Foods, Contribution to
Melanoidin Formation, Metabolism
Furan could be formed by heating of L–ascorbic acid, amino acids, reducing sugars, and fatty
acids. Nevertheless, the mechanism of formation of furan derivatives differs among each other
but all are formed by heating of one or two of the precursors. Furan and its derivatives give a
positive benefit to the sensory properties of heated food but also have toxic and in some cases
mutagenic effects. Moreover, the polymerization of furfuryl alcohol as furan derivative
contributes to the formation of the brown colour in heated foods, besides other Maillard and
caramelization reactions. During heating of food, furfuryl alcohol is formed through
degradation of quinic acid or 1,2-enediols. Furfuryl alcohol is a mutagenic compound. In acid
conditions it is able to polymerize and form aliphatic polymers that show a brown colour. In
addition, some of those furans still remain in the liver or kidney which can be metabolized
forming toxic or mutagenic compounds that bind to proteins or DNA. In this research project
it was shown that the HPLC using gradient elution with methanol and water can be used for
the identification and quantification of HMF, furfuryl alcohol, and furfural in a single run.
Coffee instant powder, ready-to-drink filter coffee, and cappuccino contain 5-
hydroxymethylfurfural (HMF), furfuryl alcohol, and furfural. Dried plums and raisins also
contain HMF and furfural. Crisp bread contains furfuryl alcohol and HMF. Besides that, goat
cheese contains furfuryl alcohol and cola beverage contains HMF. The samples analysed were
35

provided by the Norwegian Institute of Public Health investigating the exposure to these
substances in Norway. Furthermore, here we show that furfuryl alcohol polymerizes in a
model system by incubation in 1 M HCl at room temperature. Some of the reaction products
are oligomers with dimers, trimers, tetramers, and pentamers having a methylene linkage
being identified. The degree of polymerization and the amount of those furfuryl alcohol
oligomers increases with increasing reaction time. The results of this model system were used
to characterize the polymerization of furfuryl alcohol during roasting of coffee. The coffee
was roasted at 210 °C for 2, 3, 4, 5, and 6 min using a home coffee roaster. Furfuryl alcohol
and its dimer were found in coffee after 2 and 3 min of roasting reaching a maximum amount
after 4 min; probably due to further reactions the dimeric furfuryl alcohol concentration starts
to decrease after 4 min. We propose that the polymers of furfuryl alcohol contribute to the
brown colour of roasted foods. In urine, the 5-hydroxymethyl-2-furoic acid and 2-furoic acid
as metabolites can be analysed by LC/MS/MS after alkaline treatment to hydrolyse the
glycine conjugates.
Master Theses completed
Patricia Mürzl: Melatonin a physiologically active substance in foods – influence of
manufacturing on the content in products
Melatonin is a substance with antioxidant ability which occurs in the human body and in
plants. The substance, that is important for the synthesis of Melatonin, is tryptophan, which is
synthesized to serotonin. The amount of Serotonin coordinates the amount of melatonin. It´s
also named the „sleepy hormone“, because the synthesis is regulated through the light and
dark-signals in the retina. In the night, the synthesis of melatonin starts, and in the morning
the metabolism occurs. It can be synthesized too into the intestinal tract. Even high
concentrations of melatonin, can be taken up by the human body.
This master-these is about the analysing of the quantitative amount of Melatonin in foods.
For the Analysis, several extraction methods, for getting higher concentrations of the
substance, were used. Because of the low level of melatonin in foods, it was necessary to
work with a cat-ion exchanger method. Furthermore, the extraction methods with Diol- and
C18-colum were used. The detection of the substances was realized with HPLC.
The extraction methods with Diol- and C18-column were analysed with thin-layerchromatography.
For the detection of the substance, we used, a UV-detector at 264 nm.
Because of the low concentrations, the detection of the samples which were measured with
the HPLC, were detected with a fluorescence-detector at ex = 285 nm, em = 345 nm.
The analysed were mainly plant seeds, because of the ability of melatonin being an
antioxidant, the concentrations were very high. The reason why they have such great amounts
of melatonin are found could be, that they have to protect the genome of the seeds.
Brown and white mustard-seeds as well as Tomato- and grape-seeds, anise, caraway and
potatoes were analysed.
In addition, raw and cooked wild rice was analysed for melatonin. The results showed that the
preparation of foods, specially the temperature, has an influence on the amount of melatonin.
In the evening and at night milked milk was analysed too for the amount of melatonin.
Wolfgang Sindler: Strategies for mitigation of acrylamide in bread
In this work various methods for the analysis of acrylamide were used and improved, which
were based on different model systems. Compared to French fries and potato chips, cookies
do not have a high concentration of acrylamide. However they are consumed in large
quantities and therefore were used as a model system for the determination of acrylamide.
36

After purification, acrylamide was detected by liquid chromatography with different detectors
(HPLC-UV and HPLC-MS detection). On the one hand, a model system by the use of HPLC-
UV detection was analysed for acrylamide. Therefore an equimolar mixture of asparagine and
glucose was applied. The temperature and time have permanently been increased, resulting in
a rapid increase in the concentration of acrylamide.
In another model system with HPLC-MS detection (triple quadrupole mass spectrometer in
combination with electrospray ionization) acrylamide was measured with the addition of
various concentrations of asparaginase. Detection was performed in the so-called MRM mode
(Multiple reaction monitoring mode). This enzyme should prevent the formation of
acrylamide from the precursors (reducing sugars and asparagine). The investigations showed
that concentration of 900 mg Asparaginase/kg flour leads to a reduction of acrylamide of 61
%.
Another model was developed by the addition of inorganic salts (NH 4 HCO 3 , CaCl 2 ). In
comparison to the control biscuits, the addition of NH 4 HCO 3 leads to an increase, concerning
the concentration of acrylamide of 137 %, whereas CaCl 2 leads to a decrease of 89 %. Finally,
acrylamide was detected in frequently consumed foods. Here the highest concentrations were
found in chips (1506 ng/g) and coffee (756 ng/g), whereas different types of bread (rye bread,
rye breads) showed no significant differences.
Basically, it is important to find a good combination of the factors that can reduce acrylamide
(storage conditions, temperature and time control, water content and the addition of salts).
Using CaCl 2 has a pleasing effect on the reduction of acrylamide, but also causes a negative
flavour development (bitter taste component).
In summary, it can be said, that asparaginase could be used in the future in the food industry
to reduce acrylamide.
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 Institut für Ernährungsforschung, Potsdam Rehbrücke, Germany
H. Pinheiro, Instituto Superior Tecnico, Lisboa, Portugal
V. Piironen, Department of Applied Chemistry and Microbiology, Helsinki, Finland
Z. Cieserova, Food Research Institute, Bratislava, Slovakia
C. Thongkraung, Prince of Songkla University, Hatyai, Thailand
D.W. Marseno, Gadjah Mada University, Yogyakarta, Indonesia
Research project
Mitigation of acrylamide in bread by application of asparaginase
Invited Lectures
1) Murkovic, M.:
Oxidation of triacylglycerols in presence of carotenoids - Identification of oxidized
species by LC-MS. - in: Chemical Reactions in Foods VII. Prague/CZ
37

2) Murkovic, M.:
Furan Derivatives in Foods. - in: International Workshop: Towards Quality and Safe
Food. Bratislava/SL
3) Murkovic, M.:
Heat induced carcinogens in foods. - in: Viikki Food Science Seminar. Helsinki/Fin
Publications
1) Swasti, Y. R.; Murkovic, M. Characterization of the polymerization of furfuryl alcohol
during roasting of coffee. - in: Food & Function 3 (2012) 965 – 969
2) Greimel, K.; Murkovic, M. Comparison of a PCR base method and a HPLC based
method for determination of the soy content of animal feed. Ernährung/Nutrition 36
(2012) 245 – 251
3) Murkovic, M. Formation of carcinogenic substances during heating of foods. Zastita
materijala Materials protection 53 (2012) 3 – 8
4) Glatt, H.; Schneider, H.; Murkovic, M.; Monien, B. H.; Meinl, W. Hydroxymethylsubstituted
furans: mutagenicity in Salmonella typhimurium strains engineered for
expression of various human and rodent sulfotransferases. - in: Mutagenesis 27 (2012)
41 - 48
38

Course no. Title
Lectures and Laboratory Courses
Winter Semester
CHE.154_1 Biochemistry Laboratory Course I 5.33 Team
Hours Lecturer (assistant)
CHE.155 Biochemistry II 1.5 Macheroux P
CHE.191 Bioanalytics 2.25
CHE.192 Biochemistry Laboratory Course II 4 Team
MOL.832_1 Project laboratory 9 Team
MOL.842_1 Seminar for PhD students 2 Team
Hermetter A, Klimant
I
MOL.855 Molecular physiology 2 Macheroux P
MOL.863 Food Chemistry and -technology 4
Leitner E, Murkovic
M
MOL.933 Food Biotechnology 1.3 Murkovic M
MOL.954 Laboratory Project 6
MOL.957_1 Project Laboratory Bioanalytics 6 Team
Leitner E, Murkovic
M
MOL.959 Enzymatic and Microbial Food Processing 2 Murkovic M
MOL.961 Food Chemistry and -technology II 2 Murkovic M
648.003 Molecular Enzymology I 2 Macheroux P
648.005 Fundamental Chemistry 2 Hermetter A
648.007 Graduate Seminar 1 1 Team
648.009 Scientific Colloquium for Graduate Students 1 1 Team
648.057 Membrane Biophysics 2 Lohner K
648.059 Fundamentals of Pharmacology 2 Dittrich P
648.060 Membrane Mimetic Systems 1 Lohner K
648.082 Biophysical chemistry 2 Laggner P
648.087 Biophysical chemistry of lipids 2 Hermetter A
648.092 Cell biology of lipids 2 Daum G
648.100 Cell Biology 1 1 Daum G
648.214 Phys. methods in Biochemistry 2 Laggner P
648.280 Physical Methods in Biochemistry 1 Laggner P
649.023
Special chapters of food chemistry and -
technology II
2 Murkovic M
649.027 Chemical Reactions in Foods I 2 Murkovic M
649.032 Recent Trends in Food Chemistry 2 Günther K
649.033 Chemical Reactions in Foods 2 Murkovic M
39

Summer Semester
Course no. Title Hours Lecturer (assistant)
CHE.147 Biochemistry I 3.75 Macheroux P
CHE.193 Molecular biology laboratory course 3 Knaus T, Waldner I
CHE.194
Seminar for Molecular biology
laboratory course
CHE.195 Cell Biology 1.5 Daum G
MOL.406 Methods in Immunology 2 Daum G
MOL.407 Methods in Immunology 2
MOL.832_1 Project laboratory 9 Team
MOL.842_1 Seminar for PhD students 2 Team
MOL.851 Special Topics in Biochemistry 1
1 Athenstaedt K, Waldner I
MOL.876 Fluorescence technology 2 Hermetter A
MOL.877 Fluorescence technology 1.5 Hermetter A
MOL.880 Molecular enzymology 2
Binter A, Daum G, Knaus T,
Wallner S
Daum G, Harsay E, Hermetter A,
Macheroux P
Gruber K, Macheroux P,
Nidetzky B
MOL.954 Laboratory Project 6 Leitner E, Murkovic M
MOL.957_1 Project Laboratory Bioanalytics 6 Team
648.004 Molecular Enzymology II 2 Macheroux P
648.006 Introduction to Biochemistry 2 Macheroux P, Waldner I
648.008 Graduate Seminar 2 1 Team
648.010
Scientific Colloquium for Graduate
Students 2
1 Team
648.058 Membrane Biophysics 2 Lohner K
648.083 Biophysical chemistry 2 Laggner P
648.088 Biophysical chemistry of lipids 2 Hermetter A
648.093 Cell biology of lipids 2 Daum G
648.101
Structure analysis in biophysics and
material research
2 Laggner P
648.200 Cell Biology 2 1 Daum G, Harsay E
648.302 Methods in Immunology 1 Daum G
649.000
649.013
Molecular Gastronomy and Food
Processing II
Special chapters of food chemistry
and technology I
1 Murkovic M
2 Murkovic M
649.016 Chemical Reactions in Foods II 2 Murkovic M
649.017 Fish and Fish-Products 1 Murkovic M
40