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

quinone thereby catalyzing the formation of a fully reduced hydroquinone. Interestingly, those
enzymes are found in solid tumours at increased levels and thus can be used to target the tumour
cell through bioreductive activation of prodrugs. Here the identification and characterization of
Lot6p, a soluble, flavin-dependent quinone reductase from Saccharomyces cerevisiae is described.
It is shown that Lot6p is capable of reducing a variety of substrates at the expense of either NADH
or NADPH via a ping-pong bi bi reaction mechanism. Recent studies using human quinone
reductases demonstrated that this enzyme binds to the 20S proteasome and affects the lifetime of
transcription factors, such as tumour suppressor p53, by inhibiting their intracellular degradation.
It could be shown that Lot6p is physically associated with the yeast proteasome. Furthermore,
quinone reductase lacking the flavin cofactor was unable to bind to the proteasome indicating that
only the holo-protein can form a protein complex. Interaction of Lot6p with the proteasome is
independent of the redox state of the flavin. However, reduction of the FMN cofactor triggers
association of the complex with Yap4p, a transcription factor involved in the oxidative stress
response. Moreover, Yap4p was detected in the nucleus of quinone stressed yeast cells, but not in
the nucleus of non-stressed wild-type cells. This finding suggests that stress causes the release of
the transcription factor from the protein complex resulting in concomittant relocalisation to the
nucleus. Sequestration of a transcription factor by a quinone-reductase-proteasome complex
discovered here is similar to findings in mammalian cells.
Our data also reveal that the redox state of the flavin cofactor controls recruitment of the
transcription factor Yap4p to the quinone reductase-proteasome complex. This would imply that
stability and localization of Yap4p are under direct redox control of the Lot6p-proteasome
complex. Under reducing conditions (when an excess of NAD(P)H is present in the cell), Yap4p is
bound to the quinone reductase-proteasome system, virtually in a stand-by position. In the
presence of quinones (i.e., when oxidative stress is applied to the cell), Yap4p is released from the
complex and rapidly relocalizes to the nucleus where it is now able to switch on stress-related
target genes.
Andreas Winkler: Structure-function studies on berberine bridge enzyme from the California
poppy (Eschscholzia californica)
Berberine bridge enzyme catalyzes the oxidative conversion of (S)-reticuline to (S)-scoulerine by
formation of a C-C bond between the N-methyl group and the phenolic moiety of the substrate.
This reaction leads to ring closure and is unique to the biosynthesis of benzophenanthridine
alkaloids with no direct equivalent in organic chemistry. We therefore set out to characterize the
molecular mechanism by means of a functional and structural analysis of the enzyme. The
biochemical characterization of the flavin cofactor revealed that it is covalently linked to the
protein backbone via two amino acids (His-104 and Cys-166). At the time of discovery this
unusual bicovalent mode of FAD attachment was just recently identified during the structural
analysis of a distantly related enzyme. We could show that this dual mode of cofactor linkage
increases the redox potential to the highest value reported for flavoenzymes so far (+132 mV). The
analysis of mutant proteins lacking each of these linkages (H104A and C166A) confirmed their
contribution to the elevated redox potential (-100 and -80 mV difference, respectively) but in
addition also demonstrated their importance for correct positioning of the substrate in the active
site allowing efficient turnover. These conclusions were supported by the elucidation of the crystal
structures of wild type BBE as well as those of the mutant proteins H104A and C166A.
Comparing the active site architecture between these structures demonstrated that the overall
positioning of the cofactor is only slightly affected by the removal of the covalent linkages, while
surrounding amino acids respond significantly to replacement of either His-104 or Cys-166.
Especially the flexibility of Trp-165, which plays an important role during the conversion of
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