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quinone thereby catalyzing the formation of a fully reduced hydroquinone. Interestingly, those enzymes are found in solid tumours at increased levels and thus can be used to target the tumour cell through bioreductive activation of prodrugs. Here the identification and characterization of Lot6p, a soluble, flavin-dependent quinone reductase from Saccharomyces cerevisiae is described. It is shown that Lot6p is capable of reducing a variety of substrates at the expense of either NADH or NADPH via a ping-pong bi bi reaction mechanism. Recent studies using human quinone reductases demonstrated that this enzyme binds to the 20S proteasome and affects the lifetime of transcription factors, such as tumour suppressor p53, by inhibiting their intracellular degradation. It could be shown that Lot6p is physically associated with the yeast proteasome. Furthermore, quinone reductase lacking the flavin cofactor was unable to bind to the proteasome indicating that only the holo-protein can form a protein complex. Interaction of Lot6p with the proteasome is independent of the redox state of the flavin. However, reduction of the FMN cofactor triggers association of the complex with Yap4p, a transcription factor involved in the oxidative stress response. Moreover, Yap4p was detected in the nucleus of quinone stressed yeast cells, but not in the nucleus of non-stressed wild-type cells. This finding suggests that stress causes the release of the transcription factor from the protein complex resulting in concomittant relocalisation to the nucleus. Sequestration of a transcription factor by a quinone-reductase-proteasome complex discovered here is similar to findings in mammalian cells. Our data also reveal that the redox state of the flavin cofactor controls recruitment of the transcription factor Yap4p to the quinone reductase-proteasome complex. This would imply that stability and localization of Yap4p are under direct redox control of the Lot6p-proteasome complex. Under reducing conditions (when an excess of NAD(P)H is present in the cell), Yap4p is bound to the quinone reductase-proteasome system, virtually in a stand-by position. In the presence of quinones (i.e., when oxidative stress is applied to the cell), Yap4p is released from the complex and rapidly relocalizes to the nucleus where it is now able to switch on stress-related target genes. Andreas Winkler: Structure-function studies on berberine bridge enzyme from the California poppy (Eschscholzia californica) Berberine bridge enzyme catalyzes the oxidative conversion of (S)-reticuline to (S)-scoulerine by formation of a C-C bond between the N-methyl group and the phenolic moiety of the substrate. This reaction leads to ring closure and is unique to the biosynthesis of benzophenanthridine alkaloids with no direct equivalent in organic chemistry. We therefore set out to characterize the molecular mechanism by means of a functional and structural analysis of the enzyme. The biochemical characterization of the flavin cofactor revealed that it is covalently linked to the protein backbone via two amino acids (His-104 and Cys-166). At the time of discovery this unusual bicovalent mode of FAD attachment was just recently identified during the structural analysis of a distantly related enzyme. We could show that this dual mode of cofactor linkage increases the redox potential to the highest value reported for flavoenzymes so far (+132 mV). The analysis of mutant proteins lacking each of these linkages (H104A and C166A) confirmed their contribution to the elevated redox potential (-100 and -80 mV difference, respectively) but in addition also demonstrated their importance for correct positioning of the substrate in the active site allowing efficient turnover. These conclusions were supported by the elucidation of the crystal structures of wild type BBE as well as those of the mutant proteins H104A and C166A. Comparing the active site architecture between these structures demonstrated that the overall positioning of the cofactor is only slightly affected by the removal of the covalent linkages, while surrounding amino acids respond significantly to replacement of either His-104 or Cys-166. Especially the flexibility of Trp-165, which plays an important role during the conversion of 12
substrate to product, was reduced extensively. Its substantial conformational change during turnover was observed in soaking experiments with (S)-reticuline leading to complex structures with both substrate and product bound. These structures also allowed the identification of potential active site amino acids involved in the catalytic mechanism. Determination of kinetic rates for three active site protein variants identified a glutamate residue (Glu-417) to be essential for cofactor reduction and formation of the berberine bridge. Based on our findings, we proposed a catalytic mechanism for BBE that entails proton abstraction of the aromatic hydroxyl group leading to concerted C-C coupling accompanied by hydride transfer from the N-methyl group to the N5 atom of the FAD cofactor. International cooperations M. Abramic, Ruder Boskovic Institute Zagreb, Croatia F. Hollmann, Delft University of Technology, The Netherlands M. Groll, Technical University Munich, Germany T. Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A. S.-H. Liaw, National Yang-Ming University, Taipei, Taiwan B. A. Palfey, University of Michigan, Ann Arbor, U.S.A. I. Tews, Universität Heidelberg, Germany Research projects FWF P19858: “Enzymes of nikkomycin biosynthesis” FWF-Doktoratskolleg “Molecular Enzymology” WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49 family” Publications 1) Sollner, S., Schober, M., Wagner, A., Prem, A., Lorkova, L., Palfey, B. A., Groll, M., and Macheroux, P.: Quinone reductase acts as a redox switch of the 20S yeast proteasome, EMBO Reports, 2009, 10:65-70. 2) Mueller, N. J., Stueckler, C., Hall, M., Macheroux, P., Faber, K.: Epoxidation of conjugated C=C-bonds and sulfur-oxidation of thioethers mediated by NADH:FMNdependent oxidoreductases, Org. Biomol. Chem., 2009, 7:1115-1119. 3) Wallner, S., Neuwirth, M., Flicker, K., Tews, I., and Macheroux, P.: Dissection of contributions from invariant amino acids to complex formation and catalysis in the heteromeric pyridoxal-5’-phosphate synthase complex, Biochemistry, 2009, 48:1928- 1935. 4) Winkler, A., Motz, K., Riedl, Sabrina, Puhl, M., Macheroux, P., Gruber, K.: Structural roles of bicovalent flavinylation in berberine bridge enzyme, J. Biol. Chem., 2009, 284:19993-20001. 13
Page 1 and 2: Staff Members of the Institute of B
Page 3 and 4: the future direction of research. G
Page 5 and 6: 2009 in Graz under the chairmanship
Page 7 and 8: The BBE-catalysed oxidative carbon-
Page 9 and 10: Nikkomycin biosynthesis Nikkomycins
Page 11: HO R 1 H R 2 HSO 4 - Inversion H R
Page 15 and 16: Cell Biology Group Group leader: G
Page 17 and 18: Organelle association and membrane
Page 19 and 20: incomplete and did not provide the
Page 21 and 22: Diploma Thesis completed Sabine Zit
Page 23 and 24: Publications 1. Wagner, A., Grillit
Page 25 and 26: characteristics of these two TAG sy
Page 27 and 28: Biophysical chemistry group Group l
Page 29 and 30: esterases are important biocatalyst
Page 31 and 32: Doctoral Theses completed: Maria Mo
Page 33 and 34: measurement of hydrolytic activitie
Page 35 and 36: carotenoids, while 110 o C, 120 o C
Page 37 and 38: Lectures and Laboratory Courses Anl

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