Identification of the evolutionary divergence in DHDPS and DHDPR

DHDPS and DHDPR are the first two committed steps in the DAP pathway: a pathway responsible for the biosynthesis of lysine. It is only present in bacteria and plants making an important biological target. While DHDPS exists in a homotetrameric “dimer of dimers” formation in both bacteria and plants, the arrangement of monomers is different. In bacteria, the dimers face toward each other in a front to front arrangement. However, in plants, the orientation of the dimers is flipped into a back to back arrangement. An evolutionary difference is also observed in DHDPR. In bacteria, the protein exists in a homotetrameric conformation whereas in plants it has been shown to exist in a dimeric conformation. The exact reason for these differences in structure remain unclear but it is thought to due to evolutionary changes between the two organism types. In this study, a lycophyte DHDPS from Selaginella moellendorffii was found to exist in a substrate mediated equilibrium between dimer and tetramer, with no ligands bound. When the substrate pyruvate is bound to the enzyme, the equilibrium shifted to the tetrameric species. However, in the presence of the allosteric inhibitor lysine, the equilibrium was found to shift to a dimeric species in solution. This equilibrium could exist as a “missing link” in the evolution of the plant type quaternary structure of the DHDPS enzyme. Another subject of investigation was the characterisation of red, green and brown algal DHDPRs. The quaternary state of these species was found to be dimeric in nature. This corresponds to the proposed evolutionary lineage in which most of these species exist after the plant type species in the lineage. The exception to this is the green alga Chlamydomonas reinhardtii DHDPR which exists in an equilibrium between tetramer and dimer. As this organism lies in the evolutionary lineage between bacterial and plant forms, it is possible that this organisms DHDPR exists as the “divergence point” between these two species. C. reinhardtii DHDPR also contains a disulfide-dependent dimer interface. In the presence of reducing agent, the enzyme exists in an exclusively dimeric state. These evolutionary lineages could be applied to other enzyme evolution systems from the DAP pathway and beyond