Staff Members of the Institute of Biochemistry, TU Graz http://www ...

The absence of vitamin B 6 biosynthesis in humans clearly suggests that the proteins
involved are interesting new drug targets for the development of antibiotics, fungicides
and herbicides. The recent discovery of vitamin B 6 biosynthesis in the causative agent of
malaria, Plasmodium falciparum, prompted us to engage in an international
collaboration in order to characterise the chemistry and enzymology of vitamin B 6
biosynthesis in this highly pathogenic organism. Our studies will provide a detailed
understanding of the role(s) of Pdx1 and Pdx2 in P. falciparum and will enable us to
initiate a drug design/screening programme which will receive additional support from
the elucidation of the three-dimensional structures of the proteins and their active
complex (thesis project of Karlheinz Flicker).
NAD(P)H:FMN quinone reductases
Following our mechanistic and structural studies on YcnD, a nitroreductase from
Bacillus subtilis, we have cloned another NADPH-dependent flavin containing
oxidoreductase from this organism (termed YhdA). We have achieved high level
expression in E. coli BL 21 host cells and purified it to homogeneity. Steady-state
kinetic studies have shown that YhdA reduces FMN and nitro-organic compounds at the
expense of NADPH as the preferred electron donor. In parallel to the bacterial enzyme,
we have also started to investigate the properties of a homologous enzyme from
Saccharomyces cerevisiae (termed LOT6p). Despite the availability of a threedimensional
x-ray structure for YhdA (1NNI) and LOT6p (1T0I), the physiological role
of the enzyme was unclear. Our recent studies have now demonstrated that both
enzymes rapidly reduce quinones at the expense of a reduced nicotinamide cofactor.
These studies were complemented by localization studies of LOT6p in yeast, which
confirmed the cytosolic occurrence of the protein (in collaboration with Prof. Daum, TU
Graz). In order to further characterize the cellular role of LOT6p, we are now in the
process to search for protein interaction partners (thesis project of Sonja Sollner and
diploma project of Markus Schober).
Interestingly, the bacterial homolog of LOT6p, YhdA, forms stable tetramers instead of
dimers. Apparently, this higher oligomeric state gives rise to increased thermostability
(Deller et al., 2006). Inspection of the three-dimensional structure led to the
identification of four salt-bridges, which hold two dimers together (see figure).
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