Lead


Lay summary
The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) mediates the uptake and phosphorylation of carbohydrates, and is involved in signal transduction. In response to the availability of carbohydrates it modulates catabolite repression, intermediate metabolism, gene expression and chemotaxis. It is ubiquitous in bacteria but does not occur in animals and plants. The sugar specific transporters of the PTS are models to study the structure of membrane proteins and the molecular mechanism of solute transport. Uniqueness and pleiotropic function make the PTS a potential target for new antiinfectives.

The major objectives are (i) Crystallization of PTS proteins in particular of membrane spanning domains of the transporters (enzymes II of the PTS) belonging to the glucose family of PTS and characterization of their molecular function. (ii) Characterization of the bacterial dihydroxyacetone kinases, their structure, catalytic mechanism, physiological function and the transcriptional control of their expression. Dha kinases come in two flavors, ATP-dependent and PTS-dependent forms. The latter utilizes a phosphoprotein of the PTS instead of ATP as energy source. The conversion of an ATP binding site into a protein-binding site is an interesting example of functional evolution.

PTS transporters are purified from mesophilic bacteria and from the thermophilic bacterium Thermoanaerobacter tengcongensis. The proteins are then subjected to crystallization screens and their propensity for crystallization is improved by site-directed mutagenesis of critical residues (e.g. Cys), truncation of N- and C-terminal residues and whole domains. The X-ray diffraction and structure determination is done in collaboration with Prof. U. Baumann (University of Bern) and Prof. Tilman Schirmer (Biocenter Basel). The molecular mechanism of IICBGlc function is characterized using substrate-specificity mutants obtained by DNA shuffling. Novel proteins which may interact with components of the PTS are identified by screening for genes which are synthetic lethal with pts mutants.

A high resolution structure of a membrane protein catalyzing the vectorial transport of a substrate coupled to the transfer of a chemical group might reveal new insights into the structure and function of a membrane embedded protein (flexibility, induced fit to large substrate). Dihydroxyacetone is an important educt for large scale aldolase-catalyzed synthetic reactions. Insight in its metabolism and how the latter is controlled may contribute to improve the production yield of fermentation.