Based on the type of CO2 assimilation, plants can be divided in three photosynthetic types: the C3-type, the C4-type and the Crassulacean Acid Metabolism. C4 photosynthesis is an adaptation over the classical C3 pathway conferring increased photosynthetic efficiency in hot and dry environments in terms of using water, nitrogen and other mineral nutrients for the production of valuable biomass. An important step in the evolution of the C4 pathway is the recruitment of enzymes required for initial CO2 fixation and CO2 release, such as Phosphoenolpyruvate carboxylase (PEPC) and the Malic Enzyme (ME), respectively. The predecessors for these C4 enzymes are enzymes from C3 plants involved in non-photosynthetic metabolic processes. In spite of their high sequence identity the C4 type enzymes have distinctly different kinetic and regulatory properties.
We aim to elucidate the molecular basis for the changed regulatory and kinetic properties of the C4 type enzymes using a combined approach of genetics, biochemistry and structural studies. We apply structure determination by x-ray diffraction methods, site-specific amino acid substitution and kinetic studies on recombinant proteins from the C3 plant Flaveria pringlei and the C4 plant Flaveria trinervia.
Based on high resolution crystal structures of PEPC from F. pringlei and F. trinervia we have identified the molecular basis for the enhanced feedback inhibitor tolerance of the C4 enzyme which is considered an essential achievement in the evolution of the C2 PEPC from the C3 ancestor. Using our high resolution structural data we are designing mutants with improved kinetic and regulatory properties. Moreover, we are trying to improve protein stability by site-directed mutagenesis and fluorescence-based thermal stability assays. We are also interested to identify molecules that differentially interact with the C3 and C4 isoenzymes – either for selective activation or inhibition. Further targets for a more comprehensive understanding on the structural evolution of the C4 pathway are Pyruvate Phosphate Dikinase (PPDK) and Malic Enzyme (ME) from F. pringlei and F. trinervia.