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

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Biochemistry group Group leader: Peter Macheroux Secretary: Annemarie Portschy Postdoctoral Fellow: Ines Waldner-Scott PhD students: Alexandra Binter, Venugopal Gudipati, Tanja Knaus, Silvia Wallner Technicians: Eva Maria Pointner (maternity leave), Steve Stipsits, Rosemarie Trenker-El-Toukhy Alumni 2009: Sonja Sollner (PhD), Andreas Winkler (PhD), Martin Puhl (technician) General description The fundamental questions in the study of enzymes, the bio-catalysts of all living organisms, revolve around their ability to select a substrate (substrate specificity) and subject this substrate to a predetermined chemical reaction (reaction and regio-specificity). In general, only a few amino acid residues in the "active site" of an enzyme are involved in this process and hence provide the key to the processes taking place during enzyme catalysis. Therefore, the focus of our research is to achieve a deeper understanding of the functional role of amino acids in the active site of enzymes with regard to substrate-recognition and stereo- and regiospecificity of the chemical transformation. In addition, we are also interested in substrate-triggered conformational changes and how enzymes utilize cofactors (flavin, nicotinamide) to achieve catalysis. Towards these aims we employ a multidisciplinary approach encompassing kinetic, thermodynamic, spectroscopic and structural techniques. In addition, we use site-directed mutagenesis to generate mutant enzymes to probe their functional role in the mentioned processes. Furthermore, we collaborate with our partners in academia and industry to develop inhibitors for enzymes, which can yield important new insights into enzyme mechanisms and can be useful as potential lead compounds in the design of new drugs. The methods established in our laboratory comprise kinetic (stopped-flow and rapid quench analysis of enzymatic reactions), thermodynamic (isothermal titration microcalorimetry) and spectroscopic (fluorescence, circular dichroism and UV/VIS absorbance) methods. We also frequently use MALDI-TOF and ESI mass spectrometry, protein purification techniques (chromatography and electrophoresis) and modern molecular biology methods to clone and express genes of interest. A brief description of our current research projects is given below. Berberine bridge enzyme & other flavin-dependent plant oxidases Berberine bridge enzyme (BBE) is a central enzyme in the biosynthesis of berberine, a pharmaceutically important alkaloid. The enzyme possesses a covalently attached FAD moiety, which is essential for catalysis. The reaction involves the oxidation of the N-methyl group of the substrate (S)-reticuline by the enzyme-bound flavin and concomitant formation of a carbon-carbon bond (the “bridge”). The ultimate acceptor of the substrate-derived electrons is dioxygen, which reoxidizes the flavin to its resting state: 6
The BBE-catalysed oxidative carbon-carbon bond formation is a new example of the versatility of the flavin cofactor in biochemical reactions. Our goal is to understand the oxidative cyclization reaction by a biochemical and structural approach. Recently, we have developed a new expression system for BBE (using cDNA from Eschscholzia california, gold poppy) in Pichia pastoris, which produces large amounts of the protein (ca. 500 mg from a 10-L culture). The availability of suitable quantities of BBE enabled us to crystallize the protein and to solve the structure in collaboration with Prof. Karl Gruber at the Karl-Franzens University Graz (see below). Based on the three-dimensional structure of BBE, we have performed a site-directed mutagenesis program to investigate the role of amino acids present in the active site of the enzyme. In conjunction with other experiments, this has led to the formulation of a new reaction mechanism for the enzyme (thesis project of Andreas Winkler). In collaboration with Prof. Toni Kutchan at the Donald Danforth Plant Science Center in St. Louis, we have identified other plant genes that apparently encode flavin-dependent oxidases. These genes are 7
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