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To investigate the importance of these contacts for the formation of tetramers, we have generated a K109L and D137L single as well as a K109L/D137L double mutant protein. Currently, we are characterizing these muteins with respect to oligomerisation, temperature stability and catalytic activity. With this approach we hope to contribute to the understanding of protein oligomerization in general (research project of Dr. Sigrid Deller and diploma project of Alexandra Binter). Diploma thesis completed Alexandra Binter: Characterization of Bacillus subtilis NADPH:FMN oxidoreductase YhdA in respect to oligomerization, activity and thermostability. YhdA, a thermostable NADPH:FMN oxidoreductase from Bacillus subtilis, shows quinone reductase activiy and occurs as a tetramer. The two extended dimer surfaces are packed against each other by a 90º rotation of one dimer with respect to the other. This assembly is stabilized by the formation of four salt bridges between K109 and D137. To investigate the importance of those ion pair contacts, the following muteins were created and heterologously expressed in E. coli: K109L and D137 single muteins and K109L/D137L and K109/D137K double muteins. These muteins were characterized with respect to oligomerization, showing that all the muteins form dimers in solution. Only K109L/D137L is partially tetrameric in concentrated solutions and tetrameric in crystals. Consequently, the salt bridges are essential for stabilizing the dimer-dimer interface. It was shown that thermal denaturation of the dimeric muteins occurs at equally high temperatures as it does in the case of the wild-type protein. The activity as and NADPH:FMN oxidoreductase using oxygen and various quinones as substrates is reduced drastically compared to wild-type YhdA. Interestingly, the rate of the oxidative half reaction is similar to the wild-type, whereas the rate of the reductive half reaction is reduced significantly. Thus, it is concluded that binding of NADPH to the dimeric muteins is impaired. International cooperations M. Abramic, Ruder Boskovic Institute Zagreb, Croatia N. Amrhein and T. B. Fitzpatrick, Institute of Plant Sciences, ETH-Zürich, Switzerland S. Ghisla, Fachbereich Biologie, Universität Konstanz, Germany I. Jelesarov, Universität Zürich, Switzerland B. Kappes, Universitätklinikum Heidelberg, Germany T. Kutchan, Donald Danforth Plant Science Center, U.S.A. M. Groll, Technical University Munich, Germany B. A. Palfey, University of Michigan, U.S.A. I. Tews, Universität Heidelberg, Germany Research projects FWF P19858: “Enzyme der Nikkomycinbiosynthesis” FWF P17215: “Proteins involved in a novel vitamin B 6 pathway” FWF P17471: “Mechanism of chorismate synthase” EU-STRP: “Vitamin biosynthesis as target for antimalarial therapy” (VitBioMal) 12
FWF-Doktoratskolleg “Molecular Enzymology” WTZ Austria-Croatia: “Structure-function studies on metallopeptidases involved in metabolism of biologically active peptides” Invited lectures 1) Cost B22 WG2 conference on “Drug target characterization” at Schloss Seggau, Austria (27.04.2007): “Thermodynamic protein-protein interaction studies on PfPdx1 and PfPdx2 from Plasmodium falciparum”. 2) Universität Novi Sad, Serbia (04.06.2007): “Flavoenzyme catalysis in biosynthetic reactions”. Publications 1) Kommoju, P. R., Macheroux, P., and Ghisla, S.: Molecular cloning and expression of L-amino acid oxidase from the Malayan pit viper Calloselasma rhodostoma, Protein Expr Purific. 2007, 52:89-95. 2) Sollner, S., Nebauer, R., Ehammer, H., Prem, A., Deller, S., Palfey, B. A., Daum, G., and Macheroux, P.: LOT6 from Saccharomyces cerevisiae is a FMN-dependent reductase with a potential role in quinone detoxification, The FEBS Journal 2007, 274:1328-1339. 3) Rauch, G., Ehammer, H., Bornemann, S., and Macheroux, P.: Mutagenic analysis of an invariant aspartate residue in chorismate synthase supports its role as an active site base, Biochemistry 2007, 46:3768-3774. 4) Neuwirth, M., Flicker, K., Strohmeier, M., Tews, I., and Macheroux, P.: Thermodynamic characterization of the protein-protein interaction in the heteromeric Bacillus subtilis pyridoxal synthase, Biochemistry 2007, 46:5131- 5139. 5) Hall, M., Stueckler, C., Kroutil, W., Macheroux, P., and Faber, K.: Asymmetric bioreduction of activated alkenes using cloned 12-oxophytodienoate reductase isoenzymes OPR-1 and OPR-3 from Lycopersicon esculentum (tomato): A striking switch of stereopreference, Angew. Chem. Int. Ed. 2007, 46:3934-3937. 6) Pricelius, S., Held, C., Sollner, S., Deller, S., Murkovic, M., Ullrich, R., Hofrichter, M., Cavaco-Paulo, A., Macheroux, P., Guebitz, G. M.: Enzymatic reduction and oxidation of fibre-bound azo-compounds, Enzyme Microb. Techn. 2007, 40:1732-1738. 7) Winkler, A., Kutchan, T. M., and Macheroux, P.: 6-S-Cysteinylation of bi-covalently bound FAD in berberine bridge enzyme tunes the redox potential for optimal activity, J. Biol. Chem., 2007, 282:24437- 24443. 13
Page 1 and 2: Staff Members of the Institute of B
Page 3 and 4: 1973 Dr. F. PALTAUF, docent of the
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Page 31 and 32: 3) Wehinger A, Tancevski I, Schgoer

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