Strukturelle und funktionelle Analyse der Pyruvat-Phosphat Dikinase


The pyruvate phosphate dikinase (PPDK) is a versatile enzyme which catalyzes the reversible interconversion of pyruvate, inorganic phosphate (Pi) and adenosine triphosphate (ATP) to phosphoenolpyruvate (PEP), pyrophosphate (PPi) and adenosine monophosphate (AMP). PPDK is involved in the energy metabolism of several bacteria and protists as a glycolytic enzyme where it acts in the ATP-forming direction, boosting the energy efficiency of these organisms. The PEP-forming direction is used in C4 plants where the primary CO2 acceptor PEP is regenerated by PPDK. Hence, PPDK is one of the rate-limiting enzymes of C4 photosynthesis. Phosphoryl group transfer between the distant substrate binding domains is realized by the so-called swiveling domain mechanism which employs a swiveling movement of the central domain (CD). However, intermediate conformations of the CD have been unresolved so far. Similarly, the binding mode of nucleotides within the nucleotide binding domain (NBD) and the opening and closing motion of this domain were only postulated in analogy to other proteins with homologous nucleotide binding domains. A possible crosstalk between monomers in the functional homodimer or homotetramer has not been investigated so far. In the course of this work, a number of novel crystallographic structures of PPDK from the C4 plant Flaveria trinervia and the C3 plant Flaveria pringlei have been solved. These allow to derive a more detailed view on the domain movements of PPDK in its catalytic cycle. Two conformational intermediates of the CD have been trapped which show that the swiveling movement of the CD does not proceed in a linear interpolation between two extreme conformations, but instead proceeds via distinct sub-steps. Additionally, nucleotidebound (closed) and nucleotide-free (open) conformations of the NBD were identified in these structures. This hints at a possible alternate binding change mechanism employed in the functional dimeric or tetrameric assembly. Eventually a library of known kinase inhibitors was screened for inhibitory effects on PPDK. During this study, novel high efficacy inhibitors of PPDK have been identified which belong to the class of bisindolylmaleimides. A comparative in silico docking approach revealed high potential for structural adjustment of these inhibitors with the aim of an improved selectivity towards PPDK.

Heinrich-Heine-Universit├Ąt, Mathematisch-Naturwissenschaftliche Fakult├Ąt